2

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-

metric system units of length: centimeters, millimeters, micrometers, and nanometers

scopes, to include simple microscopes, compound

light microscopes, and electron microscopes

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.

USING THE METRIC SYSTEM TO EXPRESS THE

SIZES OF MICROORGANISMS
In microbiology, metric units (primarily micrometers and nanometers) are used
to express the sizes of microorganisms. The basic unit of length in the metric system, the meter (M), is equivalent to approximately 39.4 inches and is, therefore,
about 3.4 inches longer than a yard. A meter may be divided into 10 (101)
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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have been replaced by the terms micrometer (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

One meter contains

One centimeter contains
One millimeter contains

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

One micrometer contains

One nanometer contains

1
10
100
1,000
1,000,000
1,000,000,000

= 1 101
= 1 102
= 1 103
= 1 106
= 1 109

Figure 2-1. Representations of metric units of measure and numbers.

Microscopy

TABLE 2-1

Relative Sizes of Microorganisms

Organism(s)

Dimension(s)

Approximate Size (
m)

Viruses (most)

Diameter

0.010.3

Diameter
e.g., Escherichia coli (width  length)
Filaments (width)

average  1
average  1  3
1

e.g., Candida albicans (diameter)

Width

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

organisms are visible with the unaided human eye.

In the microbiology laboratory, the sizes of microorganisms are measured

using an ocular micrometer, a tiny ruler within the eyepiece (ocular) of the compound light microscope. Before it can be used to measure objects, however, the
ocular micrometer must first be calibrated, using a microscope stage measuring
device called a stage micrometer. Calibration must be performed for each of the
objective lenses to determine the distance between the marks on the ocular micrometer. The ocular micrometer can then be used to measure lengths and
widths of microbes and other objects on the specimen slide. The sizes of some
microorganisms are shown in Table 21.

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

27

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CHAPTER 2

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

Characteristics of Various Types of Microscopes

Type

Resolving
Power

Useful
Magnification

Brightfield

0.2000
m

1000

Used to observe morphology of microorganisms

such as bacteria, protozoa, fungi, and algae in
living (unstained) and nonliving (stained) state
Cannot resolve organisms less than 0.2
m,
such as spirochetes and viruses

Darkfield

0.2000
m

1000

Background is dark, and unstained organisms

can be seen
Useful for examining spirochetes
Slightly more difficult to operate than brightfield

Phase contrast

0.2000
m

1000

Can observe dense structures in living

procaryotic and eucaryotic microorganisms.

Fluorescence

0.2000
m

1000

Fluorescent dye attached to organism

Primarily a diagnostic technique
(immunofluorescence) to detect microorganisms
in cells, tissue, and clinical specimens
Training required in specimen preparation and
microscope operation

Transmission
electron
microscope
(TEM)

0.0002
m
(0.2 nm)

200,000

Specimen can be viewed on screen

Excellent resolution
Allows examination of cellular ultrastructure
and viruses
Specimen is nonliving
Image is two-dimensional

Scanning
electron
microscope
(SEM)

0.0200
m
(20 nm)

10,000

Specimen can be viewed on screen

Three-dimensional view of specimen
Useful in examining surface structure of cells
and viruses
Specimen is nonliving
Resolution is limited compared with TEM

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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Early Compound Microscopes

Hans Jansen, an optician in Middleburg, Holland, is often given credit for developing
the first compound microscope, sometime between 1590 and 1595. Although his
son, Zacharias, was only a young boy at the time, Zacharias apparently later took
over production of the Jansen microscopes. The Jansen microscopes contained two
lenses and achieved magnifications of only 3 to 9. Compound microscopes having a three-lens system were used by Marcello Malpighi in Italy and Robert Hooke
in England, both of whom published papers between 1660 and 1665 describing their
microscopic findings. Some early compound microscopes are shown in Figure 23.

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

Components of the Compound Light Microscope

Component

Location

Function
A 10 magnifying lens

Ocular lens (also known as

an eyepiece); a binocular
microscope has two
Revolving nosepiece

Above the stage

Holds the objective lenses

Objective lenses

Held in place above the stage

by the revolving nosepiece

Used to magnify objects placed

on the stage

Stage

Beneath the revolving nosepiece

Flat surface upon which the

specimen is placed

Stage adjustment knobs

(not shown in Fig. 22)

Beneath the stage

Used to move the specimen

Condenser

Beneath the stage

Contains a lens system that

focuses light onto the specimen

Iris diaphragm control arm

On the condenser

Used to adjust the amount of

light coming through the
condenser

Field diaphragm lever

Beneath the collector lens

Used to adjust the amount of

light coming through the
collector lens

Rheostat control knob

At the front of the base

Used to adjust the amount of

light being emitted by the light
bulb in the base

Condenser control knob

Beneath and behind the condenser

Used to adjust the height of the

condenser

Coarse and fine adjustment

knobs

On the arm of the microscope,

near the base

Used to focus the lenses

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

T A B L E 2 - 4 Magnifications Achieved Using the Compound

Light Microscope

Objective

Total Magnification Achieved When the Objective

Is Used in Conjunction With a 10 Ocular Lens

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

Figure 2-5. Spiral-shaped

Treponema pallidum, the etiologic
agent of syphilis, as seen by darkfield microscopy. (Koneman EW,
et al.: Color Atlas and Textbook
of Diagnostic Microbiology, 5th
ed. Philadelphia, LippincottRaven, 1997.)

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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CHAPTER 2

Figure 2-6. Modern transmission

electron microscopes are very
similar in appearance to the one
pictured here, being operated by
one of the authors (P.G.E.) of this
textbook (c. 1980). The specimen
to be examined is placed into the
column at the point indicated by
the arrow. Note the portholetype windows at the bottom of
the column, through which an image of the specimen is viewed.
The numerous knobs and dials
control the magnification, focus,
and built-in camera system. Some
of the transmission electron micrographs (TEMs) in this book
were taken using the microscope
pictured here.

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.

transmission electron micrographs (TEMs) and scanning electron micrographs

(SEMs), respectively. They are black and white images. If you ever see electron
micrographs in color, they have been artificially colorized. Figures 28, 29, and
210 show the differences in magnification and detail between electron micrographs and light photomicrographs. Refer to Table 22 for the characteristics of
various types of microscopes.

Figure 2-8. Staphylococcus aureus, as seen by light microscopy. (Original magnification,

1000.) (Photograph courtesy of W.L. Wong.)

38

CHAPTER 2

Figure 2-9. Staphylococcus aureus, as seen by transmission electron microscopy. (Original

magnification, 40,000.) (Photograph courtesy of Ray Rupel.)

Figure 2-10. The threedimensional qualities of scanning

electron microscopy clearly reveal
the corkscrew shape of cells of the
syphilis-causing spirochete,
Treponema pallidum, attached here
to rabbit testicular cells grown in
culture. (Original magnification,
8000). (Volk WA, et al.:
Essentials of Medical Microbiology,
5th ed. Philadelphia, LippincottRaven, 1996.)

Microscopy

39

REVIEW OF KEY POINTS

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.

Smaller objects can be seen using electron

microscopes, because electrons are used as
the source of illumination. The wavelength
of electrons is shorter than that of visible
light.
Transmission electron microscopes enable
scientists to see inside of cells (i.e., to see internal details). Using scanning electron microscopes, scientists are able to study surface details. The resolving power of the
transmission electron microscope is 0.2 nm,
whereas the resolving power of the scanning
electron microscope is 20 nm.
Because they are so tiny, most viruses can
only be seen using electron microscopes.
Photographs taken through the lens system
of the compound light microscope are called
photomicrographs, whereas those taken
with electron microscopes are called transmission electron micrographs and scanning
electron micrographs.

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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

2. Assume that a pin head is 1 mm

in diameter. How many spherical

bacteria (cocci), lined up side-byside, would fit across the pin

head. (Hint: Use information
from Table 21.)
a.
b.
c.
d.
e.

10
100
1000
10,000
100,000

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CHAPTER 2

3. What is the length of an average

rod-shaped bacterium (bacillus)?
a.
b.
c.
d.
e.

3 mm
3
m
3 nm
0.3 mm
0.03 mm

4. What is the total magnification

when using the high-power (highdry) objective of a compound
light microscope equipped with a
10 ocular lens?
a.
b.
c.
d.
e.

10
40
50
100
400

5. How many times better is the

resolution of the transmission
electron microscope than the resolution of the unaided human
eye?
a.
b.
c.
d.
e.

100
1000
10,000
100,000
1,000,000

6. How many times better is the

resolution of the transmission
electron microscope than the resolution of the compound light
microscope?
a.
b.
c.
d.
e.

100
1000
10,000
100,000
1,000,000

7. How many times better is the

resolution of the transmission
electron microscope than the resolution of the scanning electron
microscope?
a.
b.
c.
d.
e.

100
1000
10,000
100,000
1,000,000

8. The limiting factor of any compound light microscope (i.e., the

thing that limits its resolution to
0.2
m) is the:
a.

company from which it was

purchased.
b. number of condenser lenses
it has.
c. number of magnifying lenses
it has.
d. number of ocular lenses it
has.
e. wavelength of visible light.
9. Which of the following individuals is given credit for developing
the first compound microscope?
a.
b.
c.
d.
e.

Anton van Leeuwenhoek

Hans Jansen
Louis Pasteur
Marcello Malpighi
Robert Hooke

10. A compound light microscope

differs from a simple microscope
in that the compound light microscope contains more than one:
a.
b.
c.
d.
e.

condenser lens.
light bulb.
magnifying lens.
objective lens.
ocular lens.