USE OF THE LIGHT MICROSCOPE AND
ELECTRON MICROSCOPE. . . . . . . . . . . . . . . . . . . . . 3
INTRODUCTION
Cells are the fundamental physiological and structural units of most living organisms. Their
metabolic activities determine the physiological capabilities and tolerance of the entire organism,
while the pattern of cellular development and organization determines the structure and functions
of tissues, as well as the external morphology of the organism. Many types of cells are highly
specialized, but all have certain features in common, which may be noted by examining a series
of relatively unspecialized cells.
Cells are classified into three domains, Bacteria, Archaea and Eukarya, depending upon their
fundamental structural and biochemical differences. All members of domain Bacteria consist of
prokaryotic cells, which lack membrane-bound organelles and a defined nucleus. All members
of domain Eukarya consists of eukaryotic cells, which possess membrane organelles and nuclei.
Domain Archaea shares features of both domains bacteria and eukarya. Archaea shares the
following features with Bacteria: absence of nuclei, absence of membrane-bound organelles, and
the presence of a circular chromosome. Features of Eukarya shared with Archaea include the
absence of peptidoglycan in cell walls (not all eukaryotes have cell walls, however), presence of
more than one RNA polymerase, methionine as the first amino acid of all proteins, presence of
some introns in their genes, immunity to the antibiotics streptomycin and chloramphenicol, and
presence of histones to fold DNA into chromosomes.
It is believed that symbiotic events (by gene transfer or fusion of cells) between Bacteria and
Archaea resulted in Eukarya, with some of Bacteria developing into the organelles, mitochondria
(all eukaryotes) and chloroplasts (only some eukaryotes) of eukaryotic cells.
Domain Bacteria can be divided into several kingdoms including the gram-positive bacteria and
cyanobacteria. Division Archaea can be divided into 2 kingdoms. In contrast, domain Eukarya
is the most diverse with Kingdoms Plantae, Fungi, Animalia, and a grouping of 11 clades
formerly referred to as Kingdom Protista. The former Kingdom Protista consists mostly of
eukaryotic organisms that are unicellular and some multicellular forms such as seaweeds. The
remaining three major kingdoms-- Fungi, Plantae, and Animalia-- consist of multicellular
eukaryotes.
This exercise will provide you an opportunity to examine and study some representatives of
bacterial (domain Bacteria) and eukaryotic (domain Eukarya) cells.
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MATERIALS
live specimens of Anabaena, Euglena, Paramecium
prepared slides of bacteria (3 smear), Coprinus (mushroom), Syringia (lilac), rat liver
slides, cover slips, lens tissue, photomicrographs
A. BACTERIAL CELLS
Examine and draw the provided examples of bacterial cells. All bacterial cells are
prokaryotic, with no defined nucleus or membrane-bound organelles. However, not all
prokaryotic cells are in Domain Bacteria, others are in Domain Archaea.
Prokaryotes were the earliest organisms on the Earth. Both Bacteria and Archaea are
different from eukaryotes with respect to the following characteristics. They are very small
organisms, having a very primitive nuclear organization, with no nuclear membrane. They also
lack membranous organelles like chloroplasts and mitochondria. Archaea share some features
with eukarytoes—the presence of multiple forms of RNA polymerase, introns in some genes,
and histones. Bacteria do have some features not found in the other 2 domains of life (Archaea
and Eukarya)—peptidoglycan in the cell wall, and susceptibility to the antibiotics streptomycin
and chloramphenicol.
1.
DOMAIN BACTERIA
Examine the prepared slide of bacteria types under high power.
The slide contains samples of the three basic morphological types of bacteria. The
cocci are spherical, the bacilli are rod-like, and the spirilla are curved. What prokaryotic
characteristics do the bacteria have?
Observation of the photosynthetic bacteria of group Cyanobacteria.
These prokaryotes are autotrophic. They have chlorophyll a, which gives them the
characteristically blue-green color and is, found in the internal membranes called
photosynthetic lamellae. Some prokaryotes have the ability to fix nitrogen from the
atmosphere into biologically usable forms.
Prepare a water mount of some living Anabaena.
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Figure 3.1. Preparing a wet slide mount.
Objects that are to be viewed with the light microscope are mounted on glass slides. Live
or fresh specimens are usually mounted on the slide with a drop of water followed by a covering
to prevent desiccation and to compress the object. An edge of a glass coverslip is placed next to
the water and sample and lowered at an angle until it covers the sample. This technique
minimizes the trapping of air bubbles (Figure 3.1). Anabaena is a filamentous organism
appearing similar to a string of beads. Which prokaryotic characteristics can you see?
B.
EUKARYOTIC CELLS
1.
PROTISTA (formerly a kingdom)—unicellular eukaryotes that have plant and animallike features, but can not be classified into either kingdom.
Now that we have examined some basic prokaryotic cells, we will examine some
unicellular motile protozoan organisms. Protozoans used to be classified within the kingdom
Protista.
A. Euglena. Prepare a wet mount from the live culture, adding a drop of
Proto-SloTM to the water.
Species of Euglena are aquatic, mostly autotrophic, unicellular protozoans.
Motility is accomplished by the movement of a flagellum at the anterior end of the cell,
where its motion propels the cell forward. The flagellum is attached at the base of the
reservoir, a flask-shaped opening which collects excess water from the cell and
discharges it. Near the base of the flagellum is a darkly pigmented stigma associated with
a photoreceptor, which plays a role in light detection and probably aids in directional
movement of the cell. The obvious green color is due to the chlorophyll present in the
chloroplasts. Photosynthesis in these chloroplasts produces paramylon, a form of
polysaccharide found only in Euglenoids. Unlike true plant cells, euglenoids lack a cell
wall; therefore, the cell is fairly flexible, although a proteinaceous layer called the
pellicle, located just under the plasma membrane, prevents extreme changes in shape.
In what ways are these organisms plant-like, and in what ways are they animal-like?
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B. Paramecium. Prepare a wet mount from the live culture, adding a drop of
Proto-SloTM before adding the cover slip.
Observe their movement and structure. Unlike plant cells, the plasma membrane
of Paramecium is not surrounded by a cell wall. The surface of the cell is covered by
cilia, which provide the cell with its capability for movement or motility. The cell feeds
by sweeping food particles along the oral groove, a ciliated channel along one side of the
cell, through the mouth, and into the cytopharynx. As food accumulates at the end of the
cytopharynx, a vacuole forms around it and breaks off. Digestive enzymes are secreted
into this food vacuole by lysosomes and digestive products diffuse through the vacuolar
membrane into the cytoplasm. Eventually, the food vacuole fuses with the anal pore, a
specialized region of the cell surface toward the posterior of the cell, where it then
ruptures and expels any remaining indigestible contents. Species of Paramecium possess
two types of nuclei, a situation found in no other cells in any of the kingdoms. A
polyploid macronucleus controls the normal metabolic activities of the cell, while one or
more micronuclei control reproduction and give rise to the macronucleus.
c.
Amoeba. Prepare a wet mount from the live culture provided.
Amoebae are very simple, free-living, non-walled, protists found in both aquatic
and soil environments. At no time do they have flagella, but instead have cellular
extensions called pseudopodia, by which they move and feed. This type of creeping
movement by extending pseudopodia is called amoeboid movement. Which organelles
can you see in these cells?
2. KINGDOM FUNGI
The Kingdom Fungi is divided, on the basis of reproductive structures, into four phyla:
Zygomycota, Ascomycota, Basidiomycota, and Chytridomycota. Fungi are mostly multicellular,
aerobic, terrestrial eukaryotes. All fungi are heterotrophs living as saprophytes, parasites, or
symbionts. They have a thread-like mycelial body form composed of numerous hyphae having
chitinous walls. Reproduction occurs via non-flagellated, walled spores that are normally
released into the air for dispersal. Fungi may also reproduce vegetatively.
You are provided with a slide of the edible basidiomycote mushroom Coprinus. The
major portion of the fungus is the fruiting body, which can be divided into the base, stalk, gills,
and cap which is made up of a mass of compartmented, dikaryotic hyphae (N+N). The
vegetative, feeding mycelium, also made up mostly of dikaryotic hyphae, remains under ground.
The mushroom’s gills produce many basida with basidiospores along the outer surface.
Fertilization occurs in each basidium, followed by meiosis of the resulting zygote nucleus (2N),
producing four haploid (N) basidiospores. These germinate into new haploid hyphae. You
should be able to identify these structures on your slide.
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3.
KINGDOM PLANTAE (Fig 3.2)
You are provided with a histological slide of a cross section of a lilac foliage leaf
(Syringa). Both surfaces of the leaf are covered by a waxy cuticle which overlies a single layer
of cells called the epidermis. These cells can vary in shape and normally lack chloroplasts.
Their primary functions are to prevent desiccation and mechanical damage. Gas and water
exchange across the cuticularized epidermis is facilitated by pores or stomata which are
delineated by a pair of specialized epidermal cells called guard cells. The opening and closing
of the stomata are controlled by the turgor pressure of the guard cells, which is related to the
cellular osmotic pressure and the degree of cellular dessication.
The mesophyll (middle of the leaf) lies sandwiched between the epidermal layers and it
is composed of parenchyma cells. These cells contain chloroplasts and are the ones primarily
responsible for photosynthesis. In mature leaves, the parenchyma cells are differentiated into
two layers referred to as the palisade parenchyma and the spongy parenchyma. The palisade
layer consists of ordered cells lying adjacent to the upper epidermis. The spongy layer consists
of loosely arranged, irregular parenchyma cells adjacent to the lower epidermis. Numerous air
spaces are visible in the spongy parenchyma.
The other major structures visible in the leaf are the veins. They contain xylem and
phloem elements that are involved in the transportation of water, inorganic salts, and
photosynthetic products. In the larger veins, fibers and collenchyma may be used to strengthen
the structure.
Try to identify the different layers and cell types within the section. Within the various
cells you should be able to identify a number of cellular components. Eukaryotic plant cells have
three distinct components:
a.
The CELL WALL SYSTEM gives structural rigidity to almost all plant cells. The cell
wall is secreted outside of the living system, and consists of a primary wall formed early
in the cell's life; later, in many plant cells, a secondary wall is laid down inside the
primary wall. The walls of adjacent cells are bonded together by the middle lamella,
which is composed of a polysaccharide called pectin.
b.
The LIVING SYSTEM, or PROTOPLAST, includes the cytoplasm, organelles, and
the surrounding cell membrane, the plasma membrane (also called plasmalemma).
The most conspicuous feature within the protoplast is the nucleus, which serves
as the control center of the cell. It contains one or more discernible bodies, the nucleoli.
The cytoplasm appears almost homogeneous. In actuality, however, the cytoplasm
contains many organelles, which are minute, membraned compartments with specialized
functions. Some of the organelles are so small they can not be resolved with the light
microscope.
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c.
The VACUOLE is a large, membrane-enclosed sac within the protoplast. Its membrane,
called a tonoplast, encloses a water-based solution, referred to as cell sap. Many
different chemicals, including pigments, may be dissolved in this cell sap; which can also
contain crystallized protein
4.
KINGDOM ANIMALIA (Fig. 3.3)
You are provided with histological sections of rat liver tissue. The mammalian liver
accepts venous blood from the intestines and distributes it to the heart. The primary function of
the liver is to control and metabolize the various nutrients absorbed into the blood after a meal is
digested. The composition of the blood entering the organ via the hepatic portal vein can
therefore differ vastly from the blood that leaves to the heart. Other functions of the organ
include the secretion of bile into the alimentary tract and the degradation of red blood cells.
Histologically, the liver is a relatively simple organ being composed of one major cell
type termed the hepatocyte. These polyhedrally shaped cells are radially arranged around blood
sinuses that branch from the tributaries of the hepatic portal vein.
Examine the prepared slides of rat liver. You should be able to identify hepatocytes and
erythrocytes and white blood cells in the venous and arterial vessels of the organ. In general, the
arteries can be distinguished from the veins by a thicker wall and heavier musculature. Both
vessel types are lined by reticulo-endothelial cells.
Careful examination of the cells should reveal the presence of the nucleus with a
conspicuous nucleolus, and other organelles. Do all of the cells have nuclei? If not, why? Draw
the various cell types and write notes on the histology of each. Speculate on the major functions
performed by the various cell types and write your conclusions below your drawing.
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Figure 3.2. Plant cell
Figure 3.3. Animal Cell
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MICROSCOPICAL OBSERVATIONS OF SPECIMENS AND SLIDES
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ELECTRON MICROSCOPES
A.
THE INSTRUMENTS
The two basic kinds of electron microscopes are the Transmission Electron Microscope
(TEM) and the Scanning Electron Microscope (SEM). The transmission electron microscope is
used to study cellular structures and requires specimens which are thinner than ones used for
light microscopy in order to allow electron penetration. The scanning electron microscope is
used to study the surface of specimens and does not require thin sections.
Figure 3.4. A diagrammatic comparison of the light and electron microscopes.
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THE TRANSMISSION ELECTRON MICROSCOPE (TEM)
The first TEM was built by Knoll and Ruska in the early 1930's. Early commercial
electron microscopes were made by Siemens in Germany in 1939, and by RCA in the United
States in 1941. However, it wasn't until the 1950's, with the advent of improved tissue
preparation, that the TEM was used seriously to study biological specimens. The electron
microscope, like the light microscope, consists of a series of lenses for making magnified images
of very small objects. In many respects the TEM resembles an inverted light microscope.
However, the incident radiation providing the illumination is electrons rather than light and the
lenses are electromagnetic instead of glass. In addition, for electrons to be focused and directed
to the specimen, they must travel through a vacuum in the "column" of the electron microscope.
A diagrammatic comparison of the light and electron microscopes is shown in Figure 3.4.
The biggest advantage of the electron microscope over the light microscope is its greater
resolving power, which is obtained because electrons have shorter wavelengths than light waves.
With the light microscope the smallest particles one can resolve are those that are 0.2 micrometer
(m) or 200 nanometers (nm) apart, while the electron microscope can resolve objects that are
0.1 nm apart. Therefore, the electron microscope has a resolving power that is 2000 times greater
than the light microscope's.
THE SCANNING ELECTRON MICROSCOPE (SEM)
In 1938, Von Ardenethe built the first scanning electron microscope with two magnetic
lenses and a recording drum to record the electrons emitted from the surface of a specimen. The
first commercial SEM was built in 1965, at Cambridge, England, and its resolution was about 30
nm compared with more modem ones that have a resolution of less than 5 nm. The big advantage
of the SEM over the TEM is that large specimens requiring little preparation can be used. The
electron beam is focused into a cohesive spot that is rastered in the x- and y-planes across the
surface of a specimen, causing the release of various forms of energy, e.g., secondary electrons,
back scattered electrons, x-rays, and light. For imaging of biological specimens, the secondary
emitted electrons are most commonly used.
Scanning coils (two electromagnets) placed between the final lens and the specimen are
responsible for causing the electron beam (spot) to scan the specimen like a TV raster. The
emergent electrons are collected, processed, and eventually displayed on a CRT (Cathode Ray
Tube) screen. A diagram of the SEM is shown in Figure 3.5.
B.
SPECIMEN PREPARATION FOR THE TEM AND SEM
Although the use of electrons rather than light for imaging greatly increases resolving
power, it places several constraints on the nature of specimens which can be viewed. For
example, live specimens cannot generally be viewed because of the vacuum required to operate
the microscope.
For the TEM, ultrathin samples are obtained by sectioning plastic (epoxy resin) infiltrated
specimens on an ultramicrotome using very sharp glass or diamond knives. Once cut, the
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sections float from the knife edge into a "boat" filled with water. The sections are then picked up
from the water onto a metal grid (usually copper). Specimens on the grid are stained and dried
before being placed in the electron microscope column, whereupon a vacuum pump removes all
moisture and gases.
For the SEM, dried specimens are mounted on metal stubs and then coated with a very
thin layer of a heavy metal (usually gold or gold palladium). The metal is applied as a vapor in
an instrument called a sputter coater. The metal coating is applied primarily to increase the
secondary electron signal (contrast), and to protect the specimen from beam-induced thermal
damage. A metal stub with specimen attached is then mounted in the specimen chamber, and a
vacuum pump produces a vacuum around the specimen.
Figure 3.5. Diagram of a Scanning Electron Microscope
More recent advances in SEM design have permitted the use of hydrated specimens in
vacuums less than 0.1 atmospheres allowing the visualization of living or uncoated organisms
having rigid outer surfaces (cuticle, exoskeletons, carapaces, etc.).
C.
TOUR OF THE EM FACILITY
During the scheduled laboratory, tours of the EM facility will be conducted by its staff.
The tour will include actual observations through both a TEM and a SEM. Due to the limited
capacity of the facility, the class will be divided into groups.
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D.
CELL STRUCTURES - THE ELECTRON MICROSCOPE VIEW
OBSERVATIONS
Around the laboratory are a series of electron photomicrographs showing cell structures
and organelles at magnifications and resolutions provided by electron microscopes. These
photomicrographs are arranged according to the following outline of cell organization. Compare
the appearance of the cell structures at these magnifications with their appearance through the
light microscope.
The drawings you make should also indicate the scale or magnification.
(i)
The external surface of the cell
The outer limits of a cell may either be a cell wall or just the plasma membrane.
(ii)
Nuclear structures
In eukaryotic cells, the nuclear region is delineated by a double membrane. Note the
pores or openings in the double membrane of the nucleus; these pores facilitate communication
between the nuclear structure and the surrounding cytoplasm.
One or more nucleoli are associated with chromosomes of eukaryotic organisms. The
nucleoli are believed to be associated with particular chromosomes, and specifically with that
part of the chromosome that contains the DNA involved in production of ribosomal RNA. The
basic subunits of the ribosomes are assembled in the nucleolar area.
(iii)
The double membrane-bound organelles
a) Mitochondrion
Mitochondria are present in all eukaryotic cells and are the sites of aerobic respiration.
Note the relatively simple continuous outer membrane while the interior membrane is infolded to
form cristae. The internal compartment enclosed by the inner membrane is the matrix, and the
intermembrane space is formed between the two membrane layers. These double membrane
bound organelles are believed to have originated by an endosymbiotic event in which a
prokaryotic cell invaded or was engulfed by another cell. Mitochondria have DNA and reproduce
by a fission process similar to that of bacterial cells.
b) Chloroplast
This organelle for photosynthesis can be seen to be bounded by a double membrane. The
internal membranes or thylakoids contain the photosynthetic pigments and enzymes involved in
the capture of radiant energy and the conversion of this energy to chemical energy. The
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thylakoids form pouches with internal compartments or lumens, separated from the external
fluid, or stroma, within the organelle. An aggregation of thylakoids forms the grana,
characteristic of the chloroplasts of true plants. Like the mitochondria, chloroplasts have DNA
and are believed to have originated as components of a eukaryotic cell as a result of an
endosymbiotic association. They reproduce by fission of existing plastids.
(iv)
The single membrane-bound organelles
Some of the single membrane-bound organelles are believed to be formed by budding
from the endoplasmic reticulum, while others may have a different origin.
a) The Endo-Membrane System and Endoplasmic Reticulum
The endoplasmic reticulum is considered a major part of the endo-membrane system.
The endoplasmic reticulum is contiguous with the nuclear membrane at the nuclear pores,
extends into the cytoplasm, and probably links the plasma membrane and other membranes of
the cell. Ribosomes become attached to portions of the reticulum forming the rough endoplasmic
reticulum, while the areas devoid of ribosomes form the smooth endoplasmic reticulum.
b) Golgi Apparatus
The Golgi Apparatus, is believed to be formed from the endoplasmic reticulum. Note
the vesicles where products are concentrated and/or processed; these are believed to function in
transport and secretion.
c) Microbody and Lysosome
These organelles serve as compartments containing enzymes for specific metabolic
functions. Many of the enzymes within these organelles are lytic and are involved in the
degradation of dysfunctional organelles and materials brought into the cell by endocytosis and
phagocytosis.
(v) A non-membrane-bound organelle
Microtubules
These are hollow, rod-shaped structures made of protein subunits called tubulin. They
serve structural functions in the cytoskeleton, flagellae and in cilia, and are involved in the
movement of chromosomes during cell division.
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DRAWINGS OF ORGANELLES
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