DEPARTMENT OF MICROBIOLOGY
FEDERAL UNIVERSITY OF LAFIA
FULAFIA-MCB 228: BIOLOGICAL TECHNIQUES
(ELECTIVE)
2 CREDIT UNITS
MICROSCOPY
Introduction
One of the most important tools for studying microorganisms is the light
microscope, which uses visible light and a series of lenses to magnify objects.
These instruments are relatively easy to use and can magnify images
approximately 1,000x (1,000-fold). They are routinely used in the
laboratory to observe cell size, shape, and motility. The electron microscope,
introduced in 1931, can magnify images in excess of 100,000x, revealing
many fine details of cell structure. A major advancement came in the 1980s
with the development of the atomic force microscope, which allows scientists
to produce images of individual atoms on a surface.
1. Light Microscope
Basic principle of light microscopy
-Magnification
The modern light microscope, called a compound microscope, has multiple
magnifying lenses. The objective lens is a series of lenses housed in a tube
immediately above the object being viewed, whereas the ocular lens, or
eyepiece, is a lens close to the eye. Because the objective and ocular lenses are
used in combination, the total magnification is equal to the product of each
lens’s magnification. For example, a structure is magnified 1,000-fold when
viewed through a 10x ocular lens in series with a 100x objective lens. Most
1
, compound microscopes have a selection of objective lenses that are of different
powers—typically 4x, 10x, 40x , and 100x . The condenser lens, positioned
between the light source and the specimen, does not magnify. It focuses the
light on the specimen.
-Resolution
The usefulness of a microscope depends primarily on its resolving power,
which determines how much detail can be seen in the observed specimen.
Resolving power is a measure of the ability to distinguish two objects that are
very close together. It is defined as the minimum distance between two points
at which those points can still be observed as separate objects. A high resolving
power means that details of an image are clearer. The resolving power of a
microscope depends on the quality and type of lens, the wavelength of the
light (shorter wavelengths give better resolution), the magnification, and how
the specimen has been prepared. The maximum resolving power of the best
light microscope is 0.2um. This is sufficient to see the general morphology of
a prokaryotic cell but too low to distinguish an object the size of most viruses.
To obtain maximum resolution when using certain high power objectives such
as the 100x lens, immersion oil must be used to displace the air between the
lens and the specimen. This prevents the refraction (bending of light rays)
that occurs when light passes from glass to air. Light rays bend when they
pass from a medium of one refractive index (a measure of the relative speed
of light as it passes through the medium) to another. If refraction occurs,
some light rays will miss the relatively small openings of higher-power
objective lenses, causing the image to look fuzzy. The immersion oil prevents
refraction because it has nearly the same refractive index as glass.
-Contrast
The amount of contrast —the difference in color intensity between an object
and the background—affects how easily cells can be seen. As an example,
2
FEDERAL UNIVERSITY OF LAFIA
FULAFIA-MCB 228: BIOLOGICAL TECHNIQUES
(ELECTIVE)
2 CREDIT UNITS
MICROSCOPY
Introduction
One of the most important tools for studying microorganisms is the light
microscope, which uses visible light and a series of lenses to magnify objects.
These instruments are relatively easy to use and can magnify images
approximately 1,000x (1,000-fold). They are routinely used in the
laboratory to observe cell size, shape, and motility. The electron microscope,
introduced in 1931, can magnify images in excess of 100,000x, revealing
many fine details of cell structure. A major advancement came in the 1980s
with the development of the atomic force microscope, which allows scientists
to produce images of individual atoms on a surface.
1. Light Microscope
Basic principle of light microscopy
-Magnification
The modern light microscope, called a compound microscope, has multiple
magnifying lenses. The objective lens is a series of lenses housed in a tube
immediately above the object being viewed, whereas the ocular lens, or
eyepiece, is a lens close to the eye. Because the objective and ocular lenses are
used in combination, the total magnification is equal to the product of each
lens’s magnification. For example, a structure is magnified 1,000-fold when
viewed through a 10x ocular lens in series with a 100x objective lens. Most
1
, compound microscopes have a selection of objective lenses that are of different
powers—typically 4x, 10x, 40x , and 100x . The condenser lens, positioned
between the light source and the specimen, does not magnify. It focuses the
light on the specimen.
-Resolution
The usefulness of a microscope depends primarily on its resolving power,
which determines how much detail can be seen in the observed specimen.
Resolving power is a measure of the ability to distinguish two objects that are
very close together. It is defined as the minimum distance between two points
at which those points can still be observed as separate objects. A high resolving
power means that details of an image are clearer. The resolving power of a
microscope depends on the quality and type of lens, the wavelength of the
light (shorter wavelengths give better resolution), the magnification, and how
the specimen has been prepared. The maximum resolving power of the best
light microscope is 0.2um. This is sufficient to see the general morphology of
a prokaryotic cell but too low to distinguish an object the size of most viruses.
To obtain maximum resolution when using certain high power objectives such
as the 100x lens, immersion oil must be used to displace the air between the
lens and the specimen. This prevents the refraction (bending of light rays)
that occurs when light passes from glass to air. Light rays bend when they
pass from a medium of one refractive index (a measure of the relative speed
of light as it passes through the medium) to another. If refraction occurs,
some light rays will miss the relatively small openings of higher-power
objective lenses, causing the image to look fuzzy. The immersion oil prevents
refraction because it has nearly the same refractive index as glass.
-Contrast
The amount of contrast —the difference in color intensity between an object
and the background—affects how easily cells can be seen. As an example,
2
