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
Æ Distinguish between prokaryotic and eukaryotic cells on the basis of their structure.
Æ Become familiar with the morphology and diversity of bacteria and cyanobacteria.
Æ Identify plant cell walls, nuclei, vacuoles, chloroplasts, and leucoplasts.
Æ Compare and contrast two types of eukaryotic cells (from plants and animals) under the microscope.
Æ 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
Æ 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
Æ 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? ________________________________________
Æ Draw both specimens in your laboratory notebooks, including as much detail as possible.
Æ 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.
Æ 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.
Æ 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?
____________________________________________________________________________________
Æ 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.
Æ 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.
Æ 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.
Æ 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.
Æ 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? ______________________________
Æ 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.
Æ 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? _________
Æ 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.
Æ 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? ___________________
____________________________________________________________________________________
In light of the comparison above, can you think of another difference between plant and animal cells?
____________________________________________________________________________________
Æ 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
Æ 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.
Æ Scan the root tip for cells in prophase. Their nuclei have distinct chromosomes with chromatids wrapped
around each other.
Æ Find cells in metaphase. Can you discern the equatorial plane of the cells? What about the spindle
apparatus?
Æ Find cells in anaphase with separated chromatids. Note that there are various degrees of separation,
depending on how far advanced anaphase is.
Æ 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?
Æ 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