Download 2006S Bio153 Lab 2: Prokaryotes and Protists July 4th / July 6th

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2006S Bio153 Lab 2: Prokaryotes and Protists
July 4th / July 6th
Supporting material in the textbook: Chapters 27 & 28
Introduction: The two groups studied in today’s lab exercise represent the vast
majority of organismal diversity. Prokaryotes and protists make up all organisms
that are not fungi, animals or plants – and fungi, animals and plants represent a
very small portion of the tree of life!
Figure 1. The tree of life (based on 16s-RNA sequences).
Thus, it is a gross misrepresentation of diversity to allot these vast groups to a
single lab exercise. However, keep in mind that the great diversity in
prokaryotes is evident primarily in their array of metabolic pathways rather
than their morphology, and the internal structure of prokaryotic cells is best seen
under an electron microscope.
Part 1: Prokaryotes
Prokaryotes are an enormously diverse group of organisms with a long
evolutionary history. The most obvious differences between prokaryotic and
eukaryotic cells (i.e., the cells of protists, animals, fungi and plants) are a)
prokaryotic cells are usually much smaller than eukaryotic cells, and b)
prokaryotic cells are much simpler in structure than eukaryotic cells (they lack a
nucleus, membrane-bound organelles, etc.). There are two major
domains among prokaryotes: the Archaea and the Eubacteria (or Bacteria).
It is interesting to note that bacteria are thought to be phylogenetically closer
to eukaryotes than they are to Archaea – in other words, you are more closely
related to a bacterium such as Streptococcus than Streptococcus is to a
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methanogen! Archaea are difficult to isolate and culture; therefore, we will be
looking only at Eubacteria.
Gram staining: Species in the domain Eubacteria have cell walls that contain
a substance called peptidoglycan. Peptidoglycan is a polymer consisting of
sugars and amino acids that strengthen the bacterial cell wall. The Gram
stain attaches to peptidoglycan; thus, the amount of stain a bacterial cell holds
is a measure of how much peptidoglycan is in the cell wall. Gram-positive
bacteria have a thick layer of peptidoglycan and thus retain a lot of stain,
making them appear purple (Fig 2a; Fig 3). Gram-negative bacteria have
relatively little peptidoglycan in their cell walls, surrounded by a thin outer
membrane composed of lipopolysaccharide. This means that they retain
relatively little stain and appear pink (Fig 2b; Fig 3). Peptidoglycan accounts for
approximately 90% of the dry weight of Gram-positive bacteria, but only 10% of
the dry-weight of Gram-negative strains. Many antibiotics work by interfering
with the formation of the peptidoglycan layer.
(a)
(b)
Figure 2. Structure of Gram-positive (a) and Gram-negative (b)
bacterial cell walls.
Figure 3. Gram-positive and Gram-negative bacteria under the compound
microscope.
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Bacterial Morphology: Bacteria exist as single cells or in colonies. They do
not show true multicellularity (i.e. lack complex junctions between cells).
Bacterial cells are often named for their shape. The four most common shapes
are spherical (cocci), rod-shaped (bacilli), comma-shaped (vibrio), and
spiral shaped. The species name may also reflect the types of groupings the
individual cells make. Diplo- means that the bacterial cells occur in pairs;
Strepto- means the cells form chains, and Staphylo- means the cells form
clumps. Thus, Staphylococcus aureus (a bacterium commonly occurring on
human skin that is one of the causative agents of necrotizing fasciitis, or flesheating disease) grows as a clump of spherical cells. The cause of strep throat is
bacterium known as Streptococcus pyogenes or group A beta-hemolytic
streptococcus, which occurs as a chain of spherical cells.
Cyanobacteria: Cyanobacteria are sometimes called blue-green algae.
Although they are often blue-green, they are not algae – algae are eukaryotes
(their cells have a nucleus) and are considered the basal group of the plants.
Cyanobacteria are among the most abundant organisms on earth, and played an
extremely important role in the early history of organismal diversification. All
cyanobacteria perform oxygenic photosynthesis. Photosynthesis requires a
source of electrons, and organisms that perform oxygenic photosynthesis
(cyanobacteria and plants) split water molecules to obtain these electrons,
generating oxygen as a byproduct. Before the
evolution of eukaryotes, cyanobacteria
generated large amounts of oxygen, radically
altering the earth’s atmosphere. Although
prokaryotes lack membrane-bound organelles,
photosynthetic bacteria, such as
cyanobacteria, may be filled with tightly
packed folds of their outer membrane.
The effect of these membranes is to increase
the surface area on which photosynthesis can
take place. Anabaena is a filamentous cyanobacteria capable of nitrogen
fixation, and is common in many freshwater systems. It produces a neurotoxin
that is not fatal to humans, but at high concentrations often leads to fish kills.
The presence of Anabaena affects water quality, as it causes a bad smell and
disagreeable taste.
Exercise:
• Examine the slides of Gram-positive and Gram-negative bacteria.
Note the colour of the cells, their very small size, and the shape of the
cells.
• Examine the slide of Anabaena, noting the colour, shape and arrangement
of the cells.
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Part 2: Protists
The taxonomy of the group informally known as protists is contentious and under
constant revision, so we’ll focus on the functional differences among some
selected groups – their locomotion, their structure, and their modes of
reproduction.
The ciliates: The ciliates are unicellular protists of enormous complexity. They
are members of the lineage Alveolata (have small sacs called alveoli below the
plasma membrane). Ciliates are covered with cilia that propel them through
water and aid in feeding. Some cilia are fused into cirri, which can produce a
more forceful beat. Others are modified into trichocysts, which can be
discharged to catch prey or repel predators.
Ciliates are mostly diploid. Most ciliates have 2 types of nuclei – the
macronucleus and the micronucleus. The macronucleus has many copies of
the genome, and is transcriptionally active. The micronucleus has one copy
of the diploid genome, and is important in conjugation. Ciliates reproduce
asexually by fission, but can also participate in an exchange of genetic material
with another ciliate of an opposite mating type. This is not true sexual
reproduction, because no new individual arises from conjugation. During
conjugation, two cells join together, dissolving their cell membranes at the site of
junction and forming a cytoplasmic bridge. The macronucleus disintegrates
and the micronucleus undergoes meiosis. One haploid micronucleus from each
cell migrates across the cytoplasmic bridge and fuses with the micronucleus in
the other cell. This recombined
micronucleus gives rise to a new
macronucleus. This periodic genetic
rejuvenation is necessary for survival; cell
lines that are prevented from undergoing
conjugation eventually die out.
Paramecium has an oral groove lined
with cilia. It rotates on its axis while it
swims, sweeping food into its gullet. When
it bumps into an object, calcium channels in
the cell membrane open, and the cell rapidly depolarizes (loses electrical
charge). This reverses to direction of the cilia, and Paramecium swims
backwards. Although it feeds mostly on bacteria, it also has trichocysts which
it uses to repel predators such as Didinium.
Diatoms: Diatoms are members of the group
Stramenopila, and are a major component of
phytoplankton. They contribute greatly to
global photosynthesis and to the carbon cycle in
the oceans. Stramenopila all share a
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distinctive type of flagellum that is covered in hollow “hairs”, but in diatoms, only
reproductive cells have flagella. Diatoms have silica-based cell walls (frustules)
that fit together like the halves of a petri dish. Individual diatoms are diploid
(2N). Reproduction is mostly asexual, with daughter cells each receiving onehalf of the parent cell wall. Because the halves of the cell wall are not the same
size, one daughter cell tends to be smaller than the parent. After several
generations of asexual reproduction, average size of individuals in the population
declines, due to this unequal division. Reduction of body size in the
population can trigger sexual reproduction.
Amoebozoa: Amoeba proteus
This organism derives its name from the Greek
God Proteus, who could assume many forms. It
is a lobose amoeba, in the lineage Ameobozoa,
which includes plasmodial and cellular slime
molds. The distinctive feature of members of
this group is their ability to move through “cell
crawling”.
Amoeba proteus is a relatively large protist,
and moves using extensions of the cytoplasm called pseudopodia. The edge of
the pseudopodium is relatively stiff compared to the liquid interior: transitions
from gel (stiffer cytoplasm) to sol (more liquid cytoplasm) to gel account
for the extension of the pseudopodium. This cytoplasmic streaming is readily
visible under the compound microscope. Food is engulfed and contained within
food vacuoles. Contractile vacuoles expel excess water.
Cellular and plasmodial slime molds also exhibit “cell crawling”.
Cellular slime molds, such as Dictyostelium
discoideum, take the form of individual
amoeba when food is plentiful. When food is
scarce, these individual amoebae aggregate to
form a multicellular assembly called a
pseudoplasmodium or slug. The slug
functions as a single organism in that it has a
front and back end, moves and responds to
light and temperature gradients. The slug can
form a fruiting body with a stalk supporting one or more balls of spores
(inactive cells protected by a cell wall). These spores can develop into new
amoebae when food becomes plentiful.
Plasmodial slime molds are somewhat
different in that they form a web-like
supercell (a single cell with many diploid
nuclei). When food is scarc e, a fruiting
stalk forms and nuclei undergo meiosis
(2N → 1N) and form spores. These spores
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are dispersed and germinate to form haploid amoebae. Two haploid amoebae
fuse to form a diploid cell that eventually becomes a new supercell.
Exercise:
• Examine the protist material on display. Look at the display pictures
and prepared slides. Make a wet mount of Amoeba proteus and examine
under the compound microscope. Make diagrams and study notes.