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General Botany – BOT 105 LAB 2 Prokaryotes – Bacteria and Cyanobacteria Structure and Mitotic Division of Eukaryotic Cells

Cells are the basic units of living organisms because they perform all of the processes collectively known as “life.” With few exceptions, each living cell contains the full complement of an organism’s genetic information (DNA). In today’s laboratory, you will observe some of the basic features of prokaryotic and eukaryotic cells by exploring the diversity of organisms in Domain Bacteria, as well as the structure of eukaryotic cells and one of their modes of division, the mitotic division, or mitosis. The eukaryotic cells that you will focus on are plant cells. OBJECTIVES

Distinguish between prokaryotic and eukaryotic cells on the basis of their structure.

Become familiar with the morphology and diversity of bacteria and cyanobacteria.

Identify plant cell walls, nuclei, vacuoles, chloroplasts, and leucoplasts.

Compare and contrast two types of eukaryotic cells (from plants and animals) under the microscope.

Identify onion root tip cells in various stages of mitosis. 1. Prokaryotes – Bacteria and Cyanobacteria Domain Bacteria is an ancient lineage whose representatives were among the first organisms to evolve the capacity for photosynthesis. Today’s lab will examine two major groups, the heterotrophic bacteria and photosynthetic cyanobacteria, both of which are included in the Domain Bacteria. We study these organisms in Botany 105 because (1) they are an important, although often neglected, part of biodiversity; and (2) because they have crucial roles in all ecosystems, as decomposers (heterotrophic bacteria) and primary producers (cyanobacteria and non-photosynthetic autotrophic bacteria).

Domain Bacteria comprises twelve distinct evolutionary lineages, members of which have prokaryotic cells that are smaller and structurally simpler than all other cells we study in Botany 105. Bacterial (including cyanobacterial) cells lack a nuclear envelope, plastids, mitochondria, and other membrane-bound organelles. The cells consist of a plasma membrane and cytoplasm with ribosomes, a membrane system, and chromatin (DNA + associated proteins). Bacterial cells are enclosed by peptidoglycan cell walls and may have flagella that are structurally different from those of eukaryotes. Bacteria and cyanobacteria are unicellular, but may form simple filaments or colonies. The bacteria we will examine today are heterotrophs, while the cyanobacteria are photosynthetic autotrophs. Reproduction in both groups is predominantly asexual, by fission or budding, during which the plasma membrane and cell wall grow inward, eventually dividing the cell in half. However, portions of DNA may be exchanged between cells under certain circumstances (“sex” by conjugation). They are motile by simple flagella or by gliding, or are non-motile.

Although they are largely invisible to the human eye, pound for pound prokaryotes account for the majority of the Earth’s living biomass. Bacteria perform critical functions for the Earth’s ecology, not the least of which include (1) decomposition of organic matter, (2) fixation of atmospheric nitrogen, and (3) nutrient cycling. Cyanobacteria are important because (1) they are the largest group of prokaryotes to produce oxygen as a by-product of photosynthesis; (2) they are frequently confused with green algae; and (3) they occur as symbionts with a variety of fungi and plants that we will discuss throughout the semester. Their blue-green color comes from chlorophyll a and phycobilins. Some species have specialized cells called heterocysts for nitrogen processing and storage.

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EXERCISES

A. Heterotrophic bacteria

Place a drop of distilled water onto a microscope slide. Dip the tip of a toothpick into fresh yogurt and then stir the drop of water on the microscope slide with the tip of the toothpick. Add a drop of crystal violet staining solution and lower a coverslip over the specimen. With the 10x objective in place, find and focus on an area on the slide where there are blobs of purple. Go to the 40x objective and focus to find very small dark dots and rods.

These are heterotrophic bacteria that are feeding (by absorption) on nutrient substances in the yogurt. Their metabolic processes (nutrition, respiration) change the chemistry of the substrate upon which they are living, and this is how we get yogurt. One major criterion used for microscopic identification of bacteria is cell shape. Bacterial cells almost invariably take one of three forms: spheroid (coccus, pl. cocci), rod-shaped (bacillus, pl. bacilli), or spiral (spirilla, pl. spirilli). Reaction of the cells in certain chemicals, as well as type of motility, are also used in identification. There are two species of bacteria in yogurt: Lactobacillus bulgaricus (rod-shaped) and Streptococcus thermophilus (spherical).

cocci bacilli spirilli

B. Cyanobacteria

Prepare wet mounts of the cyanobacteria Oscillatoria and Gloeocapsa. Observe the specimens under 100x magnification. Note that Oscillatoria consists of filaments (strings) of cells, while Gloeocapsa consists of loosely arranged colonies. Observe the cyanobacteria under 400x total magnification and note their blue-green color.

Are the blue-green pigments localized, or uniformly distributed in the cells? _________________________

Are any structures visible within individual cells? ______________________________________________

Note that the filaments and colonies are surrounded by a gelatinous sheath, a transparent, mucous-like envelope secreted by the cells (to better see the sheaths you can add a drop of India ink to your wet mount: the sheath displaces the ink and its boundaries are easier to see).

Which genus has the most prominent gelatinous sheath? ________________________________________

Draw both specimens in your laboratory notebooks, including as much detail as possible.

Now make wet mounts of other cyanobacterial taxa available in lab and examine them to become familiar with other features of cyanobacterial morphology (note that each of these features is not characteristic of all cyanobacteria):

akinetes: vegetative cells transformed into thick-walled desiccation resistant ‘spores’. false branching: branches arising from breaks in a cyanobacterial filament. true branching: branches arising by lateral cell divisions of the main filament. heterocysts: enlarged, sometimes thick-walled cells which are sites of nitrogen fixation.

In your notebook make drawings of these features of bacterial cyanobacterial morphology.

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2. Structure and Mitotic Division of Eukaryotic Cells All members of kingdom Plantae are eukaryotes, organisms with cells containing a nucleus and membrane-bound organelles. Plant cells have a cellulose cell wall and a protoplast, which is delimited by the plasma membrane. The protoplast contains the cytoplasm and the nucleus, which stores a cell’s DNA in discrete stands of chromatin. The nucleus is surrounded by two membranes forming the nuclear envelope and contains one or more dense regions known as nucleoli. A variety of organelles are found in the cytoplasm, including plastids (e.g., chloroplasts, leucoplasts), mitochondria, endoplasmic reticula, and vacuoles. Plant cells are connected to neighboring cells by strands of cytoplasm extending through tiny pores known as plasmodesmata.

Eukaryotic cells reproduce asexually by a complicated process known as mitosis, and during which duplicated chromatin condenses into chromosomes that are then equally distributed into two genetically identical daughter cells. Mitosis forms the basis for growth for most organisms. We will observe the process of mitosis as it occurs in the growing tip of onion (Allium cepa) roots.

Plants grow as their cells divide, creating more cells. Normal cells go through two distinct phases during their lives: interphase and cell division. During interphase the cell duplicates its DNA in preparation for cell division. There are two distinct stages of cell division: mitosis and cytokinesis. During mitosis, the nucleus divides. Cytokinesis is the division of the cytoplasm.

There are four easily recognized stages of mitosis: prophase, metaphase, anaphase, and telophase. During prophase, the nuclear envelope breaks down, and the DNA molecules condense and coil to form chromosomes, visible as two sister chromatids connected by a kinetochore. During metaphase, the chromosomes align along the equatorial plane of the cell, and spindle fibers extend from each kinetochore to the cell’s poles. During anaphase, the spindle fibers, which are made of microtubules, move the sister chromatids toward opposite poles of the cell. During telophase, nuclear envelopes form around the two new clusters of chromosomes, which begin to uncoil. During cytokinesis, which begins during telophase, new cell walls and a middle lamella are formed. The developing cell walls and middle lamella are known as the cell plate, which starts in the center of the mother cell, between the two daughter nuclei, and expands to reach the side walls of the parent cell. The end result is two new cells that are genetically identical to the parent cell. EXERCISES

A. Eukaryotic cell structure – the plant cell

I. Remove two young leaves from the tip of a sprig of the aquatic angiosperm Elodea. Make a wet mount of the leaves so that their top surfaces are facing up. Do not let the leaves dry. Add more water when necessary. This plant was chosen because its leaves are very thin and the cells on the upper leaf surface are easy to study.

Examine the leaves under low and high magnification on your compound microscope. How does the size of this eukaryote’s cells compare with those of the cyanobacterial prokaryotes you examined earlier?

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Pick one cell on the leaf and then increase (total) magnification to 400x. You will be able to see chloroplasts, which appear as green, circular bodies in the cytoplasm. Focus slowly downward to the lower surface of the cell to clarify the intracellular distribution of the chloroplasts.

What is the three-dimensional shape of individual Elodea leaf cells? _______________________________

How are the chloroplasts distributed within the cells? ___________________________________________

How many cells thick is the leaf you are examining? ___________________________________________

Note that much of the volume of the cytoplasm is occupied by a water-filled central vacuole.

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Search for a nucleus, which may or may not be readily visible in all cells. It appears as a faint gray sphere

appressed to the cell wall. What is its function? _______________________________________________

Study the cell walls between adjacent cells. The thin line between the walls is known as the middle lamella.

Search for movement of the chloroplasts in a number of cells on the two leaves you mounted. This movement is called cytoplasmic streaming. Chloroplasts are not motile; they are moved around by the activity of the cytoplasm.

Draw a typical Elodea leaf cell illustrating the cell wall, vacuole, chloroplasts, and nucleus. Try to make your drawing at the same scale as your drawings of the cyanobacteria.

II. Prepare an epidermal peel of a red onion bulb scale. The scales are actually leaves modified for storage. Separate several leaves and select one with a dark red appearance. Snap the leaf backward and remove the thin, translucent piece of epidermis formed at the break point with forceps. Carefully prepare a wet mount of the epidermis without bruising the cells.

Note that the cells have a uniform pink appearance. This color results from the presence of water-soluble pigments called anthocyanins that are stored in the large vacuoles of cells.

Stain the onion cells by adding a drop of neutral red solution at the edge of the coverslip and allowing it to diffuse over the cells.

Set the slide aside for 5 to 10 minutes – you will use it in step IV.

III. In the meanwhile obtain a single leaf from the houseplant Zebrina. Note that the underside is a rich purple color. Anthocyanins are present in these cells, too. Grasp the leaf firmly with both hands, apply tension as if you were stretching it, and simultaneously twist the tissue as you slowly tear it apart. With a little practice, you will observe that small sections of the epidermis become separated from the rest of the leaf along the tear. Cut a little piece of epidermis and make a wet mount of it.

Scan the Zebrina epidermis under low magnification with the compound microscope. Look for needle-like crystals (raphides) that precipitate in the vacuoles. These crystals are composed of calcium oxalate, a metabolic waste product.

Examine the nucleus of one of the cells. Now search for a cell with small non-pigmented bodies clustered around the nucleus. These bodies are a type of plastid called leucoplasts. They synthesize starch,

oil, and proteins. Why might leucoplasts cluster around the nucleus? ______________________________

Draw a Zebrina cell. Include all salient features.

IV. Now examine the stained mount of onion epidermal cells that you had set aside earlier. You should be able to clearly distinguish the vacuole from the surrounding, stained cytoplasm.

Search for a red-stained nucleus and examine it under 400x. The dark objects within the nucleus are the nucleoli. They are the sites for ribosome synthesis. How many nucleoli are there per nucleus? _________

Draw an onion cell. Include all salient features.

B. Eukaryotic cell structure – Your own cells

Surprise! Although this is a botany lab, today you get to have a look at some animal cells – your own cells! People are heterotrophic eukaryotes classified in Kingdom Animalia. Therefore their cells have all features characteristic of eukaryotic cells: a nucleus and other membrane-bound organelles.

Take a clean toothpick and gently scrape the inside of your cheek. Stir the tip of the toothpick in a drop of water that you have prepared on a slide by mixing in it a small amount of methylene blue stain (too much

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of the stain would make the slide too dark and obscure the specimens). Gently lower a coverslip over the drop. Observe with the 10x objective. Cheek cells appear similar to fried eggs, their nuclei are stained dark blue, and their cytoplasm has a lighter shade of blue. Select a cell that does not have a lot of folds in it and observe with the 40x objective.

Compare the regular shape of the Elodea leaf cells and onion cells with the irregular shape of your cheek

cells. Based on this comparison, do you think animal cells have cell walls? Why? ___________________

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In light of the comparison above, can you think of another difference between plant and animal cells?

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Draw one of your cheek cells with its salient features.

Based on your observations of eukaryotic cells under the compound microscope, are bacterial cells larger

or smaller than those of eukaryotes? ________________________________________________________

C. Mitosis

Obtain a prepared slide of onion root tips. Under low magnification, locate the apex of the root. Study the cells of the root apex under 450x. All stages of mitosis should be visible. Note the presence of nucleoli in the nucleus of cells in interphase.

Scan the root tip for cells in prophase. Their nuclei have distinct chromosomes with chromatids wrapped around each other.

Find cells in metaphase. Can you discern the equatorial plane of the cells? What about the spindle apparatus?

Find cells in anaphase with separated chromatids. Note that there are various degrees of separation, depending on how far advanced anaphase is.

Look for cells with two nuclei. This is telophase. Can you detect the developing cell plate? What about nucleoli? Have any of theses cells completed cytokinesis?

Draw a cell in each of the four main stages of mitosis.

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If you are interested in cyanobacteria, this is a dichotomous key for identifying common cyanobacterial genera. Although today’s lab exercise does not include taxonomic identification of cyanobacteria, we will use such keys later on in this class to identify ferns, conifers, and basidiomycetes. 1a. Cells solitary, in irregular masses, or in colonies 2

1b. Cells arranged in filaments 6

2a. Cells solitary, or in small groups of varying number 3

2b. Cells aggregated in large numbers within a common mucilaginous sheath 4

3a. Cells single or in clusters, within distinctly concentric layers of mucilage Gloeocapsa

3b. Cells in distinct clusters within a sheath that lacks conspicuous concentric layers Chroococcus

4a. Cells arranged in ranks and files to form a flattened colony Merismopedia

4b. Colonies spherical, oval, or irregularly globular 5

5a. Colony of definite shape, often spherical, with cells evenly spaced at the periphery Coleosphaerium

5b. Colony irregular with cells crowded and scattered throughout the mucilage Microcystis

6a. Filaments aggregated in distinct, colonial masses 7

6b. Filaments solitary or in watery masses 9

7a. Filaments irregularly arranged in spherical colonies Nostoc

7b. Filaments regularly arranged in hemispherical or crustose colonies 8

8a. Filaments radiating to form hemispherical colonies; some filaments with large, cylindrical akinete as well

as terminal heterocysts Gloeotrichia

8b. Filaments parallel to one another and perpendicular to substrate, forming a crust; filaments with terminal

heterocysts but lacking akinetes Rivularia

9a. Filaments with heterocysts 10

9b. Filaments without heterocysts, all cells similar 14

10a. Heterocysts terminal, frequently adjacent to a large, cylindrical akinete Cylindrospermum

10b. Heterocysts intercalary except when associated with false branching; akinetes (if present) randomly

arranged and only slightly larger than other cells 11

11a. Filaments branched 12

11b. Filaments unbranched Anabaena

12a. Branches arising by lateral cell divisions of the main filament (true branching); filaments often

multiseriate Stigonema

12b. Branches arising from breaks in the main filament (false branching); filaments uniseriate 13

13a. False branches commonly developed in pairs Scytonema

13b. False branches usually single and associated with a break in the main filament below an intercalary

heterocyst Tolypothrix

14a. Filaments in regular spirals or coils Spirulina

14b. Filaments straight, bent, or entangled, but not coiled 15

15a. Filament with a distinct sheath that often extends beyond filaments Lyngbya

15b. Filaments lacking a distinct sheath, often exhibiting a waving motion Oscillatoria