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CHAPTER
4
Microscopy, Staining,
and Classification
Microscopy (pp. 96–105)
Microscopy refers to the use of light or electrons to magnify objects.
General Principles of Microscopy
The same general principles apply to both light and electron microscopy.
Wavelength of Radiation
Various forms of radiation differ in wavelength, which is the distance between
two corresponding parts of a wave. The human eye distinguishes different
wavelengths of light as
different colors. Moving electrons also act as waves, with wavelengths dependent
on the
voltage of an electron beam. Electron wavelengths are much smaller than those of
visible
light, and thus their use results in enhanced microscopy.
Magnification
Magnification is the apparent increase in size of an object and is indicated by
a number
followed by an “”, which is read “times.” Magnification results when a beam of
radiation
refracts (bends) as it passes through a lens.
Resolution
Resolution (also called resolving power) is the ability to distinguish between
objects that are
close together. The better the resolution, the better the ability to distinguish
two objects that
are close to one another. A principle of microscopy is that resolution distance
is dependent on
(1) the wavelength of the electromagnetic radiation and (2) the numerical
aperture of the
lens, which is its ability to gather light.
Contrast
Contrast refers to differences in intensity between two objects, or between an
object and its
background. Since most microorganisms are colorless, they are stained to
increase contrast.
The use of light that is in phase may also be used to enhance contrast.
Light Microscopy
Several classes of microscopes use various types of light to examine specimens.
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Copyright © 2014 Pearson Education, Inc.
Bright-Field Microscopes
The most common microscopes are bright-field microscopes, in which the
background
(or field) is illuminated. There are two basic types: Simple microscopes contain
a single
magnifying lens and are similar to a magnifying glass. Compound microscopes use
a series
of lenses for magnification. Light rays pass through a specimen and into an
objective lens
immediately above the object being magnified. The objective lens is really a
series of lenses,
and several objective lenses are mounted on a revolving nosepiece. An oil
immersion lens
increases the magnification and the resolution. Immersion oil is used to fill
the space between
the specimen and a lens to reduce light refraction and increase the numerical
aperture and
thus the resolution. The lenses closest to the eyes are ocular lenses, whereas
condenser
lenses lie beneath the stage of the microscope and direct light through the
slide. The total
magnification of a compound microscope is determined by multiplying the
magnification of
the objective lens by that of the ocular lens. A photograph of a microscopic
image is a micrograph.
Dark-Field Microscopes
Pale objects are best observed with dark-field microscopes, which utilize a
dark-field stop in
the condenser that prevents light from directly entering the objective lens.
Instead, light passes into the slide at an oblique angle. Only light rays
scattered by the specimen enter the objective lens and are seen, so the specimen
appears light against a dark background.
Phase Microscopes
Phase microscopes use a phase plate to retard light rays passing through the
specimen so
that they are ½ wavelength out of phase with neighboring light waves, thereby
producing
contrast. Phase-contrast microscopes produce sharply defined images in which
fine structures can be seen in living cells. Differential interference contrast
microscopes create
phase interference patterns and use prisms to split light beams into their
component colors,
giving images a dramatic three-dimensional or shadowed appearance.
Fluorescent Microscopes
Fluorescent microscopes use an ultraviolet (UV) light source to cause objects to
fluoresce.
Because UV light has a shorter wavelength than visible light, resolution is
increased. Contrast
is improved because fluorescing structures are visible against a black
background.
Confocal Microscopes
Confocal microscopes use fluorescent dyes in conjunction with ultraviolet lasers
to illuminate the fluorescent chemicals in only one thin plane of a specimen at
a time. Several images
are taken and digitized, and then computers construct three-dimensional images
of the entire
specimen.
Electron Microscopy
Because the shortest wavelength of visible light is about 400 nm, structures
closer together
than about 200 nm cannot be distinguished using light microscopy. By contrast,
electrons
traveling as waves have wavelengths between 0.01 nm and 0.001 nm; thus, their
resolving
power is much greater, and they typically magnify objects 10,000 to 100,000.
There are
two general types.
Copyright © 2014 Pearson Education, Inc.
CHAPTER 4 Microscopy, Staining, and Classification
23
Transmission Electron Microscopes
A transmission electron microscope (TEM) generates a beam of electrons that
passes
through a thinly sliced, dehydrated specimen, through magnetic fields that
manipulate and
focus the beam, and then onto a fluorescent screen that changes the electrons’
energy into
visible light.
Scanning Electron Microscopes
In a scanning electron microscope (SEM), the surface of the specimen is
visualized. The
SEM then focuses the beam of electrons back and forth across the surface of the
coated specimen, scanning it rather than penetrating it. Electrons scattered off
the surface of the specimen pass through a detector and a photomultiplier,
producing a signal displayed on a
monitor.
Probe Microscopy
Probe microscopes use miniscule electronic probes to magnify specimens more than
100,000,000. There are two types. Scanning tunneling microscopes pass a pointed
metallic
probe across and above the surface of a specimen and measure the amount of
electron flow.
They can reveal details on a specimen surface at the atomic level. Atomic force
microscopes
traverse the tip of the probe lightly on the surface of the specimen. Deflection
of a laser beam
aimed at the probe’s tip measures vertical movements translated by computer to
reveal the
specimen’s atomic topography.
Staining (pp. 105–112)
Both light and electron microscopy use staining—the coloring of specimens with
dyes—to
increase contrast.
Preparing Specimens for Staining
Preparing specimens for staining involves making a thin film of organisms—or
smear—of
the specimen on a slide, and then either passing the slide through a flame (heat
fixation) or
applying a chemical (chemical fixation) to attach the specimen firmly to the
slide.
Principles of Staining
The colored portion of a dye, known as the chromophore, typically binds to
chemicals via
covalent, ionic, or hydrogen bonds. Anionic chromophores called acidic dyes or
cationic
chromophores known as basic dyes are used to stain different portions of an
organism to aid
viewing and identification.
Simple Stains
Simple stains are composed of a single basic dye such as crystal violet and
involve no more
than soaking the smear in the dye and rinsing.
Differential Stains
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MICROBIOLOGY WITH DISEASES BY TAXONOMY, 4e
Copyright © 2014 Pearson Education, Inc.
Differential stains use more than one dye so that different cells, chemicals, or
structures can
be distinguished. The Gram stain differentiates between purple-staining Grampositive cells
and pink-staining Gram-negative cells. The procedure has four steps:
1. Flood the smear with the primary stain, crystal violet, and rinse.
2. Flood the smear with the mordant, iodine, and rinse.
3. Flood the smear with the decolorizing agent—a solution of ethanol and
acetone—and
rinse.
4. Flood the smear with the counterstain, safranin, and rinse.
The acid-fast stain is used to differentiate cells with waxy cell walls such as
cells of
Mycobacterium and Nocardia.
Endospores cannot be stained by normal techniques because their walls are
practically
impermeable to all chemicals.
Two histological stains are commonly used on tissue samples. The Gomori
methenamine
silver (GMS) stain is used to detect fungi, while the hematoxylin and eosin (HE)
stain reveals the presence of cancer cells.
Special Stains
Acidic dyes are repulsed by the negative charges on the surface of cells and
therefore do
not stain them. Such dyes that stain the background and leave the cells
colorless are called
negative (or capsule) stains. Flagellar stains bind to flagella, increase their
diameter, and
change their color, all of which increases contrast and makes them visible.
Staining for Electron Microscopy
Stains used for TEM are chemicals containing atoms of heavy metals, such as
lead, which
absorb electrons.
Classification and Identification of Microorganisms
(pp. 112–119)
Scientists sort organisms on the basis of mutual similarities into
nonoverlapping groups
called taxa. Taxonomy is the science of classification and nomenclature (rules
of naming).
Linnaeus and Taxonomic Categories
Carolus Linnaeus invented a system of taxonomy, grouping similar interbreeding
organisms
into species, species into genera, genera into families, families into orders,
orders into classes, classes into phyla, and phyla into kingdoms. He gave each
species a descriptive name
consisting of a genus name and specific epithet. This practice of naming
organisms with two
names is called binomial nomenclature.
Domains
Carl Woese proposed the existence of three taxonomic domains based on three cell
types
revealed by rRNA sequencing: Eukarya, Bacteria, and Archaea. Cells of the three
domains
differ in many other characteristics, including their cell membrane lipids,
transfer RNA
(tRNA) molecules, and sensitivity to antibiotics.
Copyright © 2014 Pearson Education, Inc.
CHAPTER 4 Microscopy, Staining, and Classification
25
Taxonomic and Identifying Characteristics
Taxonomists use one or more of five procedures to identify and classify
microorganisms:
1. Many physical characteristics are used to identify microorganisms. For
example, scientists
can usually identify protozoa, fungi, algae, and parasitic worms based solely on
their
morphology. The appearance of bacterial colonies also gives clues to help
identify
microorganisms.
2. Microbiologists also use biochemical tests, noting a particular microbe’s
ability to utilize
or produce certain chemicals.
3. Serological tests using antiserum (serum containing antibodies) can determine
whether a
microorganism produces an antigen-antibody reaction in the laboratory. In an
agglutination test, antiserum is mixed with a sample that may be antigenic:
clumping of antigen
with antibodies (agglutination) indicates the presence of the target cells.
4. Bacteriophages (or simply phages) are viruses that infect and usually destroy
bacterial
cells. Whenever a specific phage is able to infect and kill bacteria, the
resulting lack of
bacterial growth produces within the bacterial lawn a clear area called a
plaque.
5. Scientists also analyze a specimen’s nucleic acid content and sequence.
Taxonomic Keys
Microbiologists use dichotomous keys, which involve stepwise choices between
paired
characteristics, to assist in identifying microorganisms.
26
MICROBIOLOGY WITH DISEASES BY TAXONOMY, 4e
Copyright © 2014 Pearson Education, Inc.
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