EXPERIMENT 1
OBSERVATION OF PERMANENT SLIDES WITH BRIGHT FIELD MICROSCOPE
BASIS OF MICROSCOPY
Introduction
Microbiology, the branch of science that has so vastly extended and expanded our knowledge
of the living world, owes its existence to Antoni van Leeuwenhoek. In 1673, with the aid of a
crude microscope consisting of a biconcave lens enclosed in two metal plates, Leeuwenhoek
introduced the world to the existence of microbial forms of life. Over the years, microscopes
have evolved from the simple, single lens instrument of Leeuwenhoek, with a magnification
of 300*, to the present-day electron microscopes capable of magnifications greater than
250,000*.
Microscopes are designated as either light microscopes or electron microscopes. The former
use visible light or ultraviolet rays to illuminate specimens. They include brightfield,
darkfield, phase-contrast, and fluorescent instruments. Fluorescent microscopes use
ultraviolet radiations whose wavelengths are shorter than those of visible light and are not
directly perceptible to the human eye. Electron microscopes use electron beams (instead of
light rays) and magnets (instead of lenses) to observe sub microscopic particles.
Essential Features of Various Microscopes
Brightfield Microscope: This instrument contains two-lens systems for magnifying
specimens: the ocular lens in the eyepiece and the objective lens located in the nosepiece. The
specimen is illuminated by a beam of tungsten light focused on it by a substage lens called a
condenser; the result is a specimen that appears dark against a bright background. A major
limitation of this system is the absence of contrast between the specimen and the surrounding
medium, which makes it difficult to observe living cells. Therefore, most brightfield
observations are performed on nonviable, stained preparations.
Darkfield Microscope: This is similar to the ordinary light microscope; however, the
condenser system is modified so that the specimen is not illuminated directly. The condenser
directs the light obliquely so that the light is deflected or scattered from the specimen, which
then appears bright against a dark background. Living specimens may be observed more
readily with darkfield than with brightfield microscopy.
Phase-Contrast Microscope: Observation of microorganisms in an unstained state is
possible with this microscope. Its optics include special objectives and a condenser that make
visible cellular components that differ only slightly in their refractive indexes. As light is
transmitted through a specimen with a refractive index different from that of the surrounding
medium, a portion of the light is refracted (bent) due to slight variations in density and
thickness of the cellular components. The special optics convert the difference between
transmitted light and refracted rays, resulting in a significant variation in the intensity of light
and thereby producing a discernible image of the structure under study. The image appears
dark against a light background.
Fluorescent Microscope: This microscope is used most frequently to visualize specimens
that are chemically tagged with a fluorescent dye. The source of illumination is an ultraviolet
1
, (UV) light obtained from a high-pressure mercury lamp or hydrogen quartz lamp. The ocular
lens is fitted with a filter that permits the longer ultraviolet wavelengths to pass, while the
shorter wavelengths are blocked or eliminated. Ultraviolet radiations are absorbed by the
fluorescent label, and the energy is re-emitted in the form of a different wavelength in the
visible light range. The fluorescent dyes absorb at wavelengths between 230 and 350
nanometers (nm) and emit orange, yellow, or greenish light. This microscope is used
primarily for the detection of antigen-antibody reactions. Antibodies are conjugated with a
fluorescent dye that becomes excited in the presence of ultraviolet light, and the fluorescent
portion of the dye becomes visible against a black background.
Electron Microscope: This instrument provides a revolutionary method of microscopy, with
magnifications up to 1 million*. This permits visualization of sub microscopic cellular
particles as well as viral agents. In the electron microscope, the specimen is illuminated by a
beam of electrons rather than light, and the focusing is carried out by electromagnets instead
of a set of optics. These components are sealed in a tube in which a complete vacuum is
established. Transmission electron microscopes require specimens that are prepared as thin
filaments, fixed and dehydrated for the electron beam to pass freely through them. As the
electrons pass through the specimen, images are formed by directing the electrons onto
photographic film, thus making internal cellular structures visible. Scanning electron
microscopes are used for visualizing surface characteristics rather than intracellular
structures. A narrow beam of electrons scans back and forth, producing a three-dimensional
image as the electrons are reflected off the specimen’s surface. While scientists have a variety
of optical instruments with which to perform routine laboratory procedures and sophisticated
research, the compound brightfield microscope is the “workhorse” and is commonly found in
all biological laboratories. Although you should be familiar with the basic principles of
microscopy, you probably have not been exposed to this diverse array of complex and
expensive equipment. Therefore, only the compound brightfield microscope will be discussed
in depth and used to examine specimens.
BRIGHT FIELD MICROSCOPE
Components of the Microscope
Stage: A fixed platform with an opening in the centre allows the passage of light from an
illuminating source below to the lens system above the stage. This platform provides a
surface for the placement of a slide with its specimen over the central opening. In addition to
the fixed stage, most microscopes have a mechanical stage that can be moved vertically or
horizontally by means of adjustment controls. Less sophisticated microscopes have clips on
the fixed stage, and the slide must be positioned manually over the central opening.
Illumination: The light source is positioned in the base of the instrument. Some microscopes
are equipped with a built-in light source to provide direct illumination. Others are provided
with a reversible mirror that has one side flat and the other concave. An external light source,
such as a lamp, is placed in front of the mirror to direct the light upward into the lens system.
The flat side of the mirror is used for artificial light, and the concave side for sunlight.
Abbé Condenser: This component is found directly under the stage and contains two sets of
lenses that collect and concentrate light as it passes upward from the light source into the lens
2
OBSERVATION OF PERMANENT SLIDES WITH BRIGHT FIELD MICROSCOPE
BASIS OF MICROSCOPY
Introduction
Microbiology, the branch of science that has so vastly extended and expanded our knowledge
of the living world, owes its existence to Antoni van Leeuwenhoek. In 1673, with the aid of a
crude microscope consisting of a biconcave lens enclosed in two metal plates, Leeuwenhoek
introduced the world to the existence of microbial forms of life. Over the years, microscopes
have evolved from the simple, single lens instrument of Leeuwenhoek, with a magnification
of 300*, to the present-day electron microscopes capable of magnifications greater than
250,000*.
Microscopes are designated as either light microscopes or electron microscopes. The former
use visible light or ultraviolet rays to illuminate specimens. They include brightfield,
darkfield, phase-contrast, and fluorescent instruments. Fluorescent microscopes use
ultraviolet radiations whose wavelengths are shorter than those of visible light and are not
directly perceptible to the human eye. Electron microscopes use electron beams (instead of
light rays) and magnets (instead of lenses) to observe sub microscopic particles.
Essential Features of Various Microscopes
Brightfield Microscope: This instrument contains two-lens systems for magnifying
specimens: the ocular lens in the eyepiece and the objective lens located in the nosepiece. The
specimen is illuminated by a beam of tungsten light focused on it by a substage lens called a
condenser; the result is a specimen that appears dark against a bright background. A major
limitation of this system is the absence of contrast between the specimen and the surrounding
medium, which makes it difficult to observe living cells. Therefore, most brightfield
observations are performed on nonviable, stained preparations.
Darkfield Microscope: This is similar to the ordinary light microscope; however, the
condenser system is modified so that the specimen is not illuminated directly. The condenser
directs the light obliquely so that the light is deflected or scattered from the specimen, which
then appears bright against a dark background. Living specimens may be observed more
readily with darkfield than with brightfield microscopy.
Phase-Contrast Microscope: Observation of microorganisms in an unstained state is
possible with this microscope. Its optics include special objectives and a condenser that make
visible cellular components that differ only slightly in their refractive indexes. As light is
transmitted through a specimen with a refractive index different from that of the surrounding
medium, a portion of the light is refracted (bent) due to slight variations in density and
thickness of the cellular components. The special optics convert the difference between
transmitted light and refracted rays, resulting in a significant variation in the intensity of light
and thereby producing a discernible image of the structure under study. The image appears
dark against a light background.
Fluorescent Microscope: This microscope is used most frequently to visualize specimens
that are chemically tagged with a fluorescent dye. The source of illumination is an ultraviolet
1
, (UV) light obtained from a high-pressure mercury lamp or hydrogen quartz lamp. The ocular
lens is fitted with a filter that permits the longer ultraviolet wavelengths to pass, while the
shorter wavelengths are blocked or eliminated. Ultraviolet radiations are absorbed by the
fluorescent label, and the energy is re-emitted in the form of a different wavelength in the
visible light range. The fluorescent dyes absorb at wavelengths between 230 and 350
nanometers (nm) and emit orange, yellow, or greenish light. This microscope is used
primarily for the detection of antigen-antibody reactions. Antibodies are conjugated with a
fluorescent dye that becomes excited in the presence of ultraviolet light, and the fluorescent
portion of the dye becomes visible against a black background.
Electron Microscope: This instrument provides a revolutionary method of microscopy, with
magnifications up to 1 million*. This permits visualization of sub microscopic cellular
particles as well as viral agents. In the electron microscope, the specimen is illuminated by a
beam of electrons rather than light, and the focusing is carried out by electromagnets instead
of a set of optics. These components are sealed in a tube in which a complete vacuum is
established. Transmission electron microscopes require specimens that are prepared as thin
filaments, fixed and dehydrated for the electron beam to pass freely through them. As the
electrons pass through the specimen, images are formed by directing the electrons onto
photographic film, thus making internal cellular structures visible. Scanning electron
microscopes are used for visualizing surface characteristics rather than intracellular
structures. A narrow beam of electrons scans back and forth, producing a three-dimensional
image as the electrons are reflected off the specimen’s surface. While scientists have a variety
of optical instruments with which to perform routine laboratory procedures and sophisticated
research, the compound brightfield microscope is the “workhorse” and is commonly found in
all biological laboratories. Although you should be familiar with the basic principles of
microscopy, you probably have not been exposed to this diverse array of complex and
expensive equipment. Therefore, only the compound brightfield microscope will be discussed
in depth and used to examine specimens.
BRIGHT FIELD MICROSCOPE
Components of the Microscope
Stage: A fixed platform with an opening in the centre allows the passage of light from an
illuminating source below to the lens system above the stage. This platform provides a
surface for the placement of a slide with its specimen over the central opening. In addition to
the fixed stage, most microscopes have a mechanical stage that can be moved vertically or
horizontally by means of adjustment controls. Less sophisticated microscopes have clips on
the fixed stage, and the slide must be positioned manually over the central opening.
Illumination: The light source is positioned in the base of the instrument. Some microscopes
are equipped with a built-in light source to provide direct illumination. Others are provided
with a reversible mirror that has one side flat and the other concave. An external light source,
such as a lamp, is placed in front of the mirror to direct the light upward into the lens system.
The flat side of the mirror is used for artificial light, and the concave side for sunlight.
Abbé Condenser: This component is found directly under the stage and contains two sets of
lenses that collect and concentrate light as it passes upward from the light source into the lens
2
