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# 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)
25

Using this scale. human red blood cells are about 7 m in diameter. can be found in Appendix B.000.000 1 10 10.000. Most of the viruses that cause human disease range in size from about 10 to 300 nm. etc. or 100 (102) equally spaced units called centimeters. or 1 billion (109) equally spaced units called nanometers. a typical spherical bacterium (coccus.000 1. a cause of hemorrhagic fever) can be as long as 1000 nm (1 m)..000 1.1 nm). although some (e. 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. It should be noted that the old terms “micron” () and “millimicron” (m) have been replaced by the terms micrometer (m) and nanometer (nm). About seven cocci could fit side-by-side across a red blood cell. Interrelationships among these units are shown in Figure 2–1. millimeters.000.000 1 1.. although bacilli can be shorter or may form very long filaments.1 nanometer (0. Ebola virus. or 1 million (106) equally spaced units called micrometers. An angstrom (Å) is 0.000 1. pl.000 10. or 1000 (103) equally spaced units called millimeters.000. cocci) is approximately 1 m in diameter. respectively. The sizes of bacteria and protozoa are usually expressed in terms of micrometers. then 1000 cocci could be placed side-by-side on the pinhead. A typical rod-shaped bacterium (bacillus.000.26 CHAPTER 2 equally spaced units called decimeters.000 One micrometer contains One nanometer contains 1 10 100 1.000 = 1 × 101 = 1 × 102 = 1 × 103 = 1 × 106 = 1 × 109 Figure 2-1. The sizes of viruses are expressed in terms of nanometers. If the head of a pin was 1 mm (1000 m) in diameter.000 1 1.g. Formulas that can be used to convert inches into centimeters.000.. For example. pl. Representations of metric units of measure and numbers.000. .. bacilli) is about 1 m wide  3 m long.000 1.

a tiny ruler within the eyepiece (ocular) of the compound light microscope. A microscope is an optical instrument that is used to observe tiny objects. The ocular micrometer can then be used to measure lengths and widths of microbes and other objects on the specimen slide. Calibration must be performed for each of the objective lenses to determine the distance between the marks on the ocular micrometer. Candida albicans (diameter) Width 3–5 2–15 Width 10–30 Length Length Length Length Diameter Length (extended) 5–12 35–55 50–145 180–300 350–500 1000–2000 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.3 Diameter e.g. using a microscope stage measuring device called a stage micrometer. MICROSCOPES The human eye.. a magnifying glass.g. Before it can be used to measure objects. In the microbiology laboratory. The sizes of some microorganisms are shown in Table 2–1. a telescope.01–0. Escherichia coli (width  length) Filaments (width) average  1 average  1  3 1 e. often objects that cannot be seen at all with the unaided human eye. Each optical instrument 27 . the sizes of microorganisms are measured using an ocular micrometer.. a pair of binoculars. and a microscope can all be thought of as various types of optical instruments. the ocular micrometer must first be calibrated.Microscopy TABLE 2-1 Relative Sizes of Microorganisms Organism(s) Dimension(s) Approximate Size (m) Viruses (most) Diameter 0. however.

0200 m (20 nm) 10. fungi. Fluorescence 0.2000 m 1000 Background is dark.0002 m (0.000 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. tissue.28 CHAPTER 2 has a limit as to what can be seen using that instrument. such as spirochetes and viruses Darkfield 0. This limit is referred to as the resolving power or resolution of the instrument. and clinical specimens Training required in specimen preparation and microscope operation Transmission electron microscope (TEM) 0. protozoa. and algae in living (unstained) and nonliving (stained) state Cannot resolve organisms less than 0. Table 2–2 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. Resolving power is discussed in more detail later.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 .2000 m 1000 Fluorescent dye attached to organism Primarily a diagnostic technique (immunofluorescence) to detect microorganisms in cells. and unstained organisms can be seen Useful for examining spirochetes Slightly more difficult to operate than brightfield Phase contrast 0.2 m.2 nm) 200.2000 m 1000 Can observe dense structures in living procaryotic and eucaryotic microorganisms.

scientists believe that Leeuwenhoek’s simple microscopes had a maximum magnifying power of about 300 (300 times). 1996. Anton van Leeuwenhoek. Hans Jansen and his son Zacharias are often given credit for being the first. Compound Microscopes A compound microscope is a microscope that contains more than one magnifying lens. and two screws were used to adjust the position of the specimen. (B) Although his microscopes had a magnifying capability of only around 200 to 300. Images seen when using a magnifying glass usually appear about 3 to 20 times larger than the object’s actual size.) Compound light microscopes usually magnify objects about 1000 times. 2–2). Each had a tiny glass lens. 5th ed. Actually.: Essentials of Medical Microbiology. Because of his unique ability to grind glass lenses. (A and B: Volk WA. including bacteria and protozoa (Fig. Philadelphia. Leeuwenhoek was able to create remarkable drawings of different types of bacteria that he observed. It was held very close to the eye. used simple microscopes to observe many tiny objects. who was discussed in Chapter 1. (A) Leeuwenhoek’s microscopes were very simple devices. Lippincott-Raven.) Simple Microscopes A simple microscope is defined as a microscope containing only one magnifying lens. The specimen was mounted on the sharp point of a brass pin.Microscopy Figure 2-2. et al. (See the following Historical Note. mounted in a brass plate. During the late 1600s. The entire instrument was about 3 to 4 inches long. a magnifying glass could be considered a simple microscope. 29 . Although it is not known with certainty who the first person was to construct and use a compound microscope. Photographs taken through the lens system of compound microscopes are called photomicrographs.

Zacharias apparently later took over production of the Jansen microscopes. Some early compound microscopes are shown in Figure 2–3. As shown in Table 2–4. and 100 objectives. It is the wavelength of visible light (approximately 0. the more light that is needed. The condenser. both of whom published papers between 1660 and 1665 describing their microscopic findings. a drop of immersion oil must first be placed between the specimen and the objective. protozoa. Although his son. The four objectives used in most laboratory compound light microscopes are 4. the light must be properly adjusted and focused. adjusts the amount of light. A compound light microscope is shown in Figure 2–4.45 m) that limits the size of objects that can be seen using the compound light microscope. For optimal observation of the specimen. Generally. specimens are first observed using the 10 objective. and the functions of its various components are described in Table 2–3. and shapes the cone of light entering the objective. located beneath the stage. Usually. the high power or “high-dry” objective is then swung into position. sometime between 1590 and 1595. The 4 objective is rarely used in microbiology laboratories. was only a young boy at the time. because they are so tiny. the compound microscope is also referred to as a compound light microscope. Image clarity depends on the microscope’s resolving . The second magnifying lens system is in the objective. The compound light microscopes used in laboratories today contain two magnifying lens systems. The Jansen microscopes contained two lenses and achieved magnifications of only 3 to 9. However. When using the compound light microscope. which is positioned immediately above the object to be viewed. is often given credit for developing the first compound microscope. total magnification is calculated by multiplying the magnifying power of the ocular (10) by the magnifying power of the objective that you are using. focuses light onto the specimen. the higher the magnification. the immersion oil reduces the scattering of light and ensures that the light will enter the oil immersion lens. 40. Magnification alone is of little value unless the enlarged image possesses increased detail and clarity.30 CHAPTER 2 Early Compound Microscopes Hans Jansen. Zacharias. it usually has a magnifying power of 10. To use the oil-immersion objective. 10. the oil-immersion objective (total magnification  1000) must be used to study bacteria. objects cannot be seen if they are smaller than half of the wavelength of visible light. and other large microorganisms. Within the eyepiece or ocular is a lens called the ocular lens. Holland. an optician in Middleburg. Compound microscopes having a three-lens system were used by Marcello Malpighi in Italy and Robert Hooke in England. Because visible light (from a built-in light bulb) is used as the source of illumination. Once the specimen is in focus. This lens can be used to study algae.

A Leeuwenhoek microscope (center). surrounded by examples of early compound light microscopes.) 31 .Microscopy Figure 2-3. (Not to scale.

In practical terms. A modern compound light microscope. Thus. That distance between them. this means that objects can be examined with the compound micro- . 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.2 mm. is referred to as the resolving power of the optical instrument. they are so close together that they appear to be one object).. which is the ability of the lens system to distinguish between two adjacent objects. Knowing the resolving power of an optical instrument also defines the smallest object that can be seen with that instrument.2 mm in diameter. where they cease to be seen as separate objects.e. power (or resolution). If two objects are moved closer and closer together. For example. the resolving power of the unaided human eye is approximately 0. The resolving power of the compound light microscope is approximately 1000 times better than the resolving power of the unaided human eye.32 CHAPTER 2 Figure 2-4. the unaided human eye is unable to see objects smaller than 0.

Additional magnifying lenses could be added to the compound light microscope. 2–2) 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. as long 33 .2 m in diameter.Microscopy Table 2–3 Components of the Compound Light Microscope Component Location Function A 10 magnifying lens Ocular lens (also known as an eyepiece). we can see objects down to about 0. 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. Using a compound light microscope. As stated earlier. but this would not increase the resolving power. 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.

Fluorescence microscopy is often used in immunology laboratories to demonstrate that antibodies stained with a fluorescent dye have combined with specific antigens. laboratory technologists do not really see the treponemes—they see the light being reflected off the bacteria. Because the light refracted by living cells is different from the light refracted by the surrounding medium. called Treponema pallidum—cannot be seen with a brightfield microscope because it is thinner than 0.34 CHAPTER 2 T A B L E 2 . is beneath the resolving power of the compound light microscope. The etiologic (causative) agent of syphilis—a spiral-shaped bacterium.2 m and. much in the same way that you can “see” dust particles in a beam of sunlight.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. and that light is easily seen against the dark background (Fig. however. that microscope is sometimes referred to as a brightfield microscope. and the microscope has been converted into a darkfield microscope. Other types of compound microscopes include phase contrast microscopes and fluorescence microscopes. In the clinical microbiology laboratory. Increasing magnification without increasing the resolving power is called empty magnification. Because objects are observed against a bright background (or “bright field”) when using a compound light microscope.) . Fluorescence microscopes contain a built-in ultraviolet (UV) light source. contrast is increased. 2–5). (Immunodiagnostic procedures are described in Chapter 16. Phase contrast microscopes can be used to observe unstained living microorganisms. causing them to glow against a dark background. objects smaller than half of the wavelength of visible light cannot be seen. Dust particles are actually beneath the resolving power of the unaided eye and. With the darkfield microscope. this is a type of immunodiagnostic procedure. therefore. these substances emit a longer wavelength light. illuminated objects are seen against a dark background (or “dark field”). If the regularly used condenser is replaced with what is known as a darkfield condenser. cannot really be seen. When UV light strikes certain dyes and pigments. What you see in the beam is sunlight being reflected off the dust particles. Treponema pallidum can be seen using a darkfield microscope. darkfield microscopy is routinely used to diagnose primary syphilis (the initial stage of syphilis). and the organisms are more easily seen. It does no good to increase magnification without increasing resolving power. therefore.

they would be unable to survive in the vacuum created within the electron microscope. 2–6) has a very tall column. the etiologic agent of syphilis.g. such as rabies and smallpox viruses.. a magnification is achieved that is about 1000 times greater than the maximum magnification achieved using a compound light microscope. 1997. Organisms are killed during the specimen processing procedures. using a transmission electron microscope. It should be noted that electron microscopes cannot be used to observe living organisms.Microscopy Figure 2-5. et al. Even very tiny microbes (e. were known to exist. The first transmission electron microscopes were developed during the late 1920s and 35 . viruses) can be observed using a transmission electron microscope. An image of the specimen is produced on a phosphor-coated screen at the bottom of the microscope’s column. as seen by darkfield microscopy. and some are blocked. There are two types of electron microscopes: transmission electron microscopes and scanning electron microscopes. Because thin sections of cells are examined.000 times shorter—electron microscopes have a much greater resolving power than compound light microscopes. Spiral-shaped Treponema pallidum. Even if they were not. transmission electron microscopy enables scientists to study the internal structure of cells.: Color Atlas and Textbook of Diagnostic Microbiology. LippincottRaven.) Electron Microscopes Although extremely small infectious agents. The object can be magnified up to approximately 1 million times. at the top of which an electron gun fires a beam of electrons downward. Because the wavelength of electrons traveling in a vacuum is much shorter than the wavelength of visible light—about 100. A transmission electron microscope (Fig. they could not be seen until the electron microscope was developed. 5th ed. Special staining procedures are used to increase contrast between different parts of the cell. Electron microscopes use an electron beam as a source of illumination and magnets to focus the beam. Philadelphia. some of the electrons are transmitted through the specimen. Thus. When an extremely thin specimen (less than 1 m thick) is placed into the electron beam. (Koneman EW.

1980). Modern transmission electron microscopes are very similar in appearance to the one pictured here. The photographs taken using transmission and scanning electron microscopes are called . Electrons that bounce off the surface of the specimen are captured by detectors. focus. Some of the transmission electron micrographs (TEMs) in this book were taken using the microscope pictured here. 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. and an image of the specimen appears on a monitor. Scanning electron microscopes became available during the late 1960s. Both types of electron microscopes have built-in camera systems. 2–7) has a shorter column and. being operated by one of the authors (P. A scanning electron microscope (Fig.2 nm). The numerous knobs and dials control the magnification. through which an image of the specimen is viewed. instead of being placed into the electron beam. surface detail). Scanning electron microscopes are used to observe the outer surfaces of specimens (i.) of this textbook (c.e.E. and built-in camera system. but it was not until the early 1950s that electron microscopes began to be used routinely to study cells. Note the portholetype windows at the bottom of the column. The specimen to be examined is placed into the column at the point indicated by the arrow. it is still possible to observe extremely tiny objects using a scanning electron microscope.36 CHAPTER 2 Figure 2-6. the specimen is placed at the bottom of the column. early 1930s..G.

as seen by light microscopy. 1000. and 2–10 show the differences in magnification and detail between electron micrographs and light photomicrographs.) . Wong. (Original magnification. Figures 2–8. they have been artificially colorized. 2–9.) (Photograph courtesy of W. They are black and white images.Microscopy 37 Figure 2-7.L. transmission electron micrographs (TEMs) and scanning electron micrographs (SEMs). Scanning electron microscope. Figure 2-8. respectively. Staphylococcus aureus. Refer to Table 2–2 for the characteristics of various types of microscopes. If you ever see electron micrographs in color.

: Essentials of Medical Microbiology.000. 40.38 CHAPTER 2 Figure 2-9.) (Photograph courtesy of Ray Rupel.) . et al. The threedimensional qualities of scanning electron microscopy clearly reveal the corkscrew shape of cells of the syphilis-causing spirochete.) Figure 2-10. (Original magnification. Treponema pallidum. attached here to rabbit testicular cells grown in culture. (Original magnification. Philadelphia. 5th ed. Staphylococcus aureus. 1996. 8000). (Volk WA. as seen by transmission electron microscopy. LippincottRaven.

000 100. Because they are so tiny. or 1 billion nanometers. Assume that a pin head is 1 mm in diameter.000 . e.2 m. (Hint: Use information from Table 2–1. most viruses can only be seen using electron microscopes.000. 100 centimeters. to see internal details). 100 1000 10. because electrons are used as the source of illumination. The development of simple and compound light microscopes enabled the discovery and visualization of microorganisms. How many spherical bacteria (cocci).000 1. scientists are able to study surface details. Using scanning electron microscopes. Photographs taken through the lens system of the compound light microscope are called photomicrographs. lined up side-byside. A millimeter is equivalent to how many nanometers? a. whereas the resolving power of the scanning electron microscope is 20 nm. Simple microscopes have only one magnifying lens. 1000 millimeters.Microscopy 39 REVIEW OF KEY POINTS ■ ■ ■ ■ A meter (M) can be divided into 10 decimeters. ■ ■ ■ ■ Smaller objects can be seen using electron microscopes. The limiting factor of compound light microscopes is the type of illumination being used. The wavelength of electrons is shorter than that of visible light.) a. b. The sizes of bacteria and protozoa are expressed in micrometers (m).000 2. ON THE WEB—http://connection. b. e. c. 1.000 100.. Transmission electron microscopes enable scientists to see inside of cells (i. The metric system is used to describe the sizes of microorganisms. Because visible light is used as the source of illumination. answer the following multiple choice questions. objects that are smaller than half the wavelength of visible light cannot be seen.2 nm. would fit across the pin head. 10 100 1000 10. whereas those taken with electron microscopes are called transmission electron micrographs and scanning electron micrographs. The resolving power (resolution) of the compound light microscope is 0. whereas compound microscopes have more than one magnifying lens. d. whereas the sizes of viruses are expressed in nanometers (nm). d.e.lww.com/go/burton7e ■ ■ Critical Thinking Additional Self-Assessment Exercises SELF-ASSESSMENT EXERCISES After you have read Chapter 2. c. The resolving power of the transmission electron microscope is 0. 1 million micrometers.

000 1. What is the total magnification when using the high-power (highdry) objective of a compound light microscope equipped with a 10 ocular lens? a. 3 mm 3 m 3 nm 0.. A compound light microscope differs from a simple microscope in that the compound light microscope contains more than one: a. ocular lens. d. b. d. d. How many times better is the resolution of the transmission electron microscope than the resolution of the scanning electron microscope? a. What is the length of an average rod-shaped bacterium (bacillus)? a. magnifying lens. e. c. d. d. b. light bulb. 100 1000 10.000 7. e.3 mm 0.000 100. d. e. c. e. b. e. e. d. c. e. c. 10 40 50 100 400 5.40 CHAPTER 2 3. 9. b. c. condenser lens. 100 1000 10. d. number of condenser lenses it has. c.2 m) is the: a.000 1. Which of the following individuals is given credit for developing the first compound microscope? a. b. b.000 100. number of magnifying lenses it has. b. the thing that limits its resolution to 0. c. c.000. e. Anton van Leeuwenhoek Hans Jansen Louis Pasteur Marcello Malpighi Robert Hooke 10.000. .000 8.e.000. b. wavelength of visible light. The limiting factor of any compound light microscope (i. How many times better is the resolution of the transmission electron microscope than the resolution of the compound light microscope? a. How many times better is the resolution of the transmission electron microscope than the resolution of the unaided human eye? a.000 1.000 6.000 100. number of ocular lenses it has. 100 1000 10.03 mm 4. objective lens. company from which it was purchased.