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Microscopy
INTRODUCTION
USING THE METRIC SYSTEM
TO EXPRESS THE SIZES OF
MICROORGANISMS
MICROSCOPES
Simple Microscopes
Compound Microscopes
Electron Microscopes
LEARNING OBJECTIVES
AFTER STUDYING THIS CHAPTER, YOU SHOULD
State the metric units used to express the sizes of
BE ABLE TO:
bacteria, protozoa, and viruses
Explain the interrelationships among the following Compare and contrast the various types of micro-
INTRODUCTION
By definition, microorganisms are tiny organisms. But, how tiny are they?
Generally, some type of microscope is required to see them; thus, microorganisms are said to be microscopic. Various types of microscopes are discussed in
this chapter. The metric system will be discussed first, however, because metric
system units of length are used to express the sizes of microorganisms and the
resolving power of optical instruments.
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CHAPTER 2
equally spaced units called decimeters; or 100 (102) equally spaced units called
centimeters; or 1000 (103) equally spaced units called millimeters; or 1 million
(106) equally spaced units called micrometers; or 1 billion (109) equally spaced
units called nanometers. Interrelationships among these units are shown in
Figure 21. Formulas that can be used to convert inches into centimeters, millimeters, etc., can be found in Appendix B.
It should be noted that the old terms micron () and millimicron (m)
have been replaced by the terms micrometer (m) and nanometer (nm), respectively. An angstrom () is 0.1 nanometer (0.1 nm). Using this scale, human red
blood cells are about 7 m in diameter.
The sizes of bacteria and protozoa are usually expressed in terms of micrometers. For example, a typical spherical bacterium (coccus; pl., cocci) is approximately 1 m in diameter. About seven cocci could fit side-by-side across a
red blood cell. If the head of a pin was 1 mm (1000 m) in diameter, then 1000
cocci could be placed side-by-side on the pinhead. A typical rod-shaped bacterium (bacillus; pl., bacilli) is about 1 m wide 3 m long, although bacilli
can be shorter or may form very long filaments. The sizes of viruses are expressed in terms of nanometers. Most of the viruses that cause human disease
range in size from about 10 to 300 nm, although some (e.g., Ebola virus, a cause
of hemorrhagic fever) can be as long as 1000 nm (1 m). Some very large protozoa reach a length of 2000 m (2 mm).
1 meter
Centimeters
Millimeters
Micrometers
Nanometers
100
1,000
1,000,000
1,000,000,000
10
10,000
10,000,000
1,000
1,000,000
1,000
1
10
100
1,000
1,000,000
1,000,000,000
= 1 101
= 1 102
= 1 103
= 1 106
= 1 109
Microscopy
TABLE 2-1
Organism(s)
Dimension(s)
Viruses (most)
Diameter
0.010.3
Diameter
e.g., Escherichia coli (width length)
Filaments (width)
average 1
average 1 3
1
35
215
Width
1030
Length
Length
Length
Length
Diameter
Length (extended)
512
3555
50145
180300
350500
10002000
Bacteria
Cocci (spherical bacteria)
Bacilli (rod-shaped bacteria)
Fungi
Yeasts
Septate hyphae (hyphae with
cross-walls)
Aseptate hyphae (hyphae without
cross-walls)
Pond water protozoa
Chlamydomonas
Euglena
Vorticella
Paramecium
Volvoxa
Stentora
aThese
MICROSCOPES
The human eye, a telescope, a pair of binoculars, a magnifying glass, and a microscope can all be thought of as various types of optical instruments. A microscope is an optical instrument that is used to observe tiny objects, often objects
that cannot be seen at all with the unaided human eye. Each optical instrument
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has a limit as to what can be seen using that instrument. This limit is referred to
as the resolving power or resolution of the instrument. Resolving power is discussed in more detail later. Table 22 contains the resolving powers for various
optical instruments.
TABLE 2-2
Type
Resolving
Power
Useful
Magnification
Brightfield
0.2000 m
1000
Darkfield
0.2000 m
1000
Phase contrast
0.2000 m
1000
Fluorescence
0.2000 m
1000
Transmission
electron
microscope
(TEM)
0.0002 m
(0.2 nm)
200,000
Scanning
electron
microscope
(SEM)
0.0200 m
(20 nm)
10,000
Characteristics
Microscopy
Figure 2-2. (A) Leeuwenhoeks microscopes were very simple devices. Each had a tiny glass
lens, mounted in a brass plate. The specimen was mounted on the sharp point of a brass pin.
and two screws were used to adjust the position of the specimen. The entire instrument was
about 3 to 4 inches long. It was held very close to the eye. (B) Although his microscopes had
a magnifying capability of only around 200 to 300, Leeuwenhoek was able to create remarkable drawings of different types of bacteria that he observed. (A and B: Volk WA, et al.:
Essentials of Medical Microbiology, 5th ed. Philadelphia, Lippincott-Raven, 1996.)
Simple Microscopes
A simple microscope is defined as a microscope containing only one magnifying
lens. Actually, a magnifying glass could be considered a simple microscope.
Images seen when using a magnifying glass usually appear about 3 to 20 times
larger than the objects actual size. During the late 1600s, Anton van
Leeuwenhoek, who was discussed in Chapter 1, used simple microscopes to observe many tiny objects, including bacteria and protozoa (Fig. 22). Because of
his unique ability to grind glass lenses, scientists believe that Leeuwenhoeks
simple microscopes had a maximum magnifying power of about 300 (300
times).
Compound Microscopes
A compound microscope is a microscope that contains more than one magnifying lens. Although it is not known with certainty who the first person was to construct and use a compound microscope, Hans Jansen and his son Zacharias are
often given credit for being the first. (See the following Historical Note.)
Compound light microscopes usually magnify objects about 1000 times.
Photographs taken through the lens system of compound microscopes are called
photomicrographs.
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Because visible light (from a built-in light bulb) is used as the source of illumination, the compound microscope is also referred to as a compound light microscope. It is the wavelength of visible light (approximately 0.45 m) that limits the size of objects that can be seen using the compound light microscope.
When using the compound light microscope, objects cannot be seen if they are
smaller than half of the wavelength of visible light. A compound light microscope is shown in Figure 24, and the functions of its various components are described in Table 23.
The compound light microscopes used in laboratories today contain two
magnifying lens systems. Within the eyepiece or ocular is a lens called the ocular lens; it usually has a magnifying power of 10. The second magnifying lens
system is in the objective, which is positioned immediately above the object to
be viewed. The four objectives used in most laboratory compound light microscopes are 4, 10, 40, and 100 objectives. As shown in Table 24, total
magnification is calculated by multiplying the magnifying power of the ocular
(10) by the magnifying power of the objective that you are using.
The 4 objective is rarely used in microbiology laboratories. Usually, specimens are first observed using the 10 objective. Once the specimen is in focus,
the high power or high-dry objective is then swung into position. This lens can
be used to study algae, protozoa, and other large microorganisms. However, the
oil-immersion objective (total magnification 1000) must be used to study
bacteria, because they are so tiny. To use the oil-immersion objective, a drop of
immersion oil must first be placed between the specimen and the objective; the
immersion oil reduces the scattering of light and ensures that the light will enter
the oil immersion lens.
For optimal observation of the specimen, the light must be properly adjusted and focused. The condenser, located beneath the stage, focuses light onto
the specimen, adjusts the amount of light, and shapes the cone of light entering
the objective. Generally, the higher the magnification, the more light that is
needed.
Magnification alone is of little value unless the enlarged image possesses increased detail and clarity. Image clarity depends on the microscopes resolving
Microscopy
Figure 2-3. A Leeuwenhoek microscope (center), surrounded by examples of early compound light microscopes. (Not to scale.)
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CHAPTER 2
Figure 2-4.
A modern
compound light
microscope.
power (or resolution), which is the ability of the lens system to distinguish between two adjacent objects. If two objects are moved closer and closer together,
there comes a point when the objects are so close together that the lens system
can no longer resolve them as two separate objects (i.e., they are so close together
that they appear to be one object). That distance between them, where they cease
to be seen as separate objects, is referred to as the resolving power of the optical
instrument. Knowing the resolving power of an optical instrument also defines
the smallest object that can be seen with that instrument. For example, the resolving power of the unaided human eye is approximately 0.2 mm. Thus, the unaided human eye is unable to see objects smaller than 0.2 mm in diameter.
The resolving power of the compound light microscope is approximately
1000 times better than the resolving power of the unaided human eye. In practical terms, this means that objects can be examined with the compound micro-
Microscopy
Table 23
Component
Location
Function
A 10 magnifying lens
Objective lenses
Stage
Condenser
On the condenser
scope that are as much as 1000 times smaller than the smallest objects that can
be seen with the unaided human eye. Using a compound light microscope, we
can see objects down to about 0.2 m in diameter.
Additional magnifying lenses could be added to the compound light microscope, but this would not increase the resolving power. As stated earlier, as long
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CHAPTER 2
Objective
4 (scanning objective)
10 (low-power objective)
40 (high-dry objective)
100 (oil immersion objective)
40
100
400
1000
as visible light is used as the source of illumination, objects smaller than half of
the wavelength of visible light cannot be seen. Increasing magnification without
increasing the resolving power is called empty magnification. It does no good to
increase magnification without increasing resolving power.
Because objects are observed against a bright background (or bright field)
when using a compound light microscope, that microscope is sometimes referred
to as a brightfield microscope. If the regularly used condenser is replaced with
what is known as a darkfield condenser, illuminated objects are seen against a
dark background (or dark field), and the microscope has been converted into
a darkfield microscope. In the clinical microbiology laboratory, darkfield microscopy is routinely used to diagnose primary syphilis (the initial stage of
syphilis). The etiologic (causative) agent of syphilisa spiral-shaped bacterium,
called Treponema pallidumcannot be seen with a brightfield microscope because it is thinner than 0.2 m and, therefore, is beneath the resolving power of
the compound light microscope. Treponema pallidum can be seen using a darkfield microscope, however, much in the same way that you can see dust particles in a beam of sunlight. Dust particles are actually beneath the resolving
power of the unaided eye and, therefore, cannot really be seen. What you see in
the beam is sunlight being reflected off the dust particles. With the darkfield microscope, laboratory technologists do not really see the treponemesthey see
the light being reflected off the bacteria, and that light is easily seen against the
dark background (Fig. 25).
Other types of compound microscopes include phase contrast microscopes
and fluorescence microscopes. Phase contrast microscopes can be used to observe unstained living microorganisms. Because the light refracted by living cells
is different from the light refracted by the surrounding medium, contrast is increased, and the organisms are more easily seen. Fluorescence microscopes contain a built-in ultraviolet (UV) light source. When UV light strikes certain dyes
and pigments, these substances emit a longer wavelength light, causing them to
glow against a dark background. Fluorescence microscopy is often used in immunology laboratories to demonstrate that antibodies stained with a fluorescent
dye have combined with specific antigens; this is a type of immunodiagnostic
procedure. (Immunodiagnostic procedures are described in Chapter 16.)
Microscopy
Electron Microscopes
Although extremely small infectious agents, such as rabies and smallpox viruses,
were known to exist, they could not be seen until the electron microscope was
developed. It should be noted that electron microscopes cannot be used to observe living organisms. Organisms are killed during the specimen processing
procedures. Even if they were not, they would be unable to survive in the vacuum created within the electron microscope.
Electron microscopes use an electron beam as a source of illumination and
magnets to focus the beam. Because the wavelength of electrons traveling in a
vacuum is much shorter than the wavelength of visible lightabout 100,000
times shorterelectron microscopes have a much greater resolving power than
compound light microscopes. There are two types of electron microscopes:
transmission electron microscopes and scanning electron microscopes.
A transmission electron microscope (Fig. 26) has a very tall column, at the
top of which an electron gun fires a beam of electrons downward. When an extremely thin specimen (less than 1 m thick) is placed into the electron beam,
some of the electrons are transmitted through the specimen, and some are
blocked. An image of the specimen is produced on a phosphor-coated screen at
the bottom of the microscopes column. The object can be magnified up to approximately 1 million times. Thus, using a transmission electron microscope, a
magnification is achieved that is about 1000 times greater than the maximum
magnification achieved using a compound light microscope. Even very tiny microbes (e.g., viruses) can be observed using a transmission electron microscope.
Because thin sections of cells are examined, transmission electron microscopy
enables scientists to study the internal structure of cells. Special staining procedures are used to increase contrast between different parts of the cell. The first
transmission electron microscopes were developed during the late 1920s and
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early 1930s, but it was not until the early 1950s that electron microscopes began
to be used routinely to study cells.
A scanning electron microscope (Fig. 27) has a shorter column and, instead
of being placed into the electron beam, the specimen is placed at the bottom of
the column. Electrons that bounce off the surface of the specimen are captured
by detectors, and an image of the specimen appears on a monitor. Scanning electron microscopes are used to observe the outer surfaces of specimens (i.e., surface detail). Although the resolving power of scanning electron microscopes
(about 20 nm) is not quite as good as the resolving power of transmission electron microscopes (about 0.2 nm), it is still possible to observe extremely tiny objects using a scanning electron microscope. Scanning electron microscopes became available during the late 1960s.
Both types of electron microscopes have built-in camera systems. The photographs taken using transmission and scanning electron microscopes are called
Microscopy
37
Figure 2-7.
Scanning electron
microscope.
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CHAPTER 2
Microscopy
39
A meter (M) can be divided into 10 decimeters, 100 centimeters, 1000 millimeters, 1 million micrometers, or 1 billion nanometers.
The metric system is used to describe the
sizes of microorganisms. The sizes of bacteria and protozoa are expressed in micrometers (m), whereas the sizes of viruses are
expressed in nanometers (nm).
The development of simple and compound
light microscopes enabled the discovery and
visualization of microorganisms. Simple microscopes have only one magnifying lens,
whereas compound microscopes have more
than one magnifying lens.
The limiting factor of compound light microscopes is the type of illumination being used.
Because visible light is used as the source of
illumination, objects that are smaller than
half the wavelength of visible light cannot be
seen. The resolving power (resolution) of the
compound light microscope is 0.2 m.
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Critical Thinking
Additional Self-Assessment Exercises
SELF-ASSESSMENT EXERCISES
After you have read Chapter 2, answer the following multiple choice questions.
1. A millimeter is equivalent to how
many nanometers?
a.
b.
c.
d.
e.
100
1000
10,000
100,000
1,000,000
10
100
1000
10,000
100,000
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CHAPTER 2
3 mm
3 m
3 nm
0.3 mm
0.03 mm
10
40
50
100
400
100
1000
10,000
100,000
1,000,000
100
1000
10,000
100,000
1,000,000
100
1000
10,000
100,000
1,000,000
condenser lens.
light bulb.
magnifying lens.
objective lens.
ocular lens.