INTRODUCTIONUSING THE METRIC SYSTEM

TO EXPRESS THE SIZES OFMICROORGANISMS

MICROSCOPESSimple MicroscopesCompound MicroscopesElectron Microscopes

AFTER STUDYING THIS CHAPTER, YOU SHOULD

BE ABLE TO:

■ Explain the interrelationships among the followingmetric system units of length: centimeters, mil-limeters, micrometers, and nanometers

■ State the metric units used to express the sizes ofbacteria, protozoa, and viruses

■ Compare and contrast the various types of micro-scopes, to include simple microscopes, compoundlight microscopes, and electron microscopes

LEARNING OBJECTIVES

Microscopy22

25

INTRODUCTION

By definition, microorganisms are tiny organisms. But, how tiny are they?Generally, some type of microscope is required to see them; thus, microorgan-isms are said to be microscopic. Various types of microscopes are discussed inthis chapter. The metric system will be discussed first, however, because metricsystem units of length are used to express the sizes of microorganisms and theresolving power of optical instruments.

USING THE METRIC SYSTEM TO EXPRESS THESIZES OF MICROORGANISMS

In microbiology, metric units (primarily micrometers and nanometers) are usedto express the sizes of microorganisms. The basic unit of length in the metric sys-tem, 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)

equally spaced units called decimeters; or 100 (102) equally spaced units calledcentimeters; or 1000 (103) equally spaced units called millimeters; or 1 million(106) equally spaced units called micrometers; or 1 billion (109) equally spacedunits called nanometers. Interrelationships among these units are shown inFigure 2–1. Formulas that can be used to convert inches into centimeters, mil-limeters, 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It should be noted that the old terms “micron” (�) and “millimicron” (m�)have been replaced by the terms micrometer (�m) and nanometer (nm), respec-tively. An angstrom (Å) is 0.1 nanometer (0.1 nm). Using this scale, human redblood cells are about 7 �m in diameter.

The sizes of bacteria and protozoa are usually expressed in terms of mi-crometers. For example, a typical spherical bacterium (coccus; pl., cocci) is ap-proximately 1 �m in diameter. About seven cocci could fit side-by-side across ared blood cell. If the head of a pin was 1 mm (1000 �m) in diameter, then 1000cocci could be placed side-by-side on the pinhead. A typical rod-shaped bac-terium (bacillus; pl., bacilli) is about 1 �m wide � 3 �m long, although bacillican be shorter or may form very long filaments. The sizes of viruses are ex-pressed in terms of nanometers. Most of the viruses that cause human diseaserange in size from about 10 to 300 nm, although some (e.g., Ebola virus, a causeof hemorrhagic fever) can be as long as 1000 nm (1 �m). Some very large pro-tozoa reach a length of 2000 �m (2 mm).

26 CHAPTER 2

1 meter

Centimeters

One meter contains

101001,0001,000,0001,000,000,000

= 1 × 101

= 1 × 102

= 1 × 103

= 1 × 106

= 1 × 109

One centimeter contains

One millimeter contains

One micrometer contains

One nanometer contains

Millimeters Micrometers Nanometers

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

1 10 10,000 10,000,000

1 1,000 1,000,000

1 1,000

1

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

In the microbiology laboratory, the sizes of microorganisms are measuredusing an ocular micrometer, a tiny ruler within the eyepiece (ocular) of the com-pound light microscope. Before it can be used to measure objects, however, theocular micrometer must first be calibrated, using a microscope stage measuringdevice called a stage micrometer. Calibration must be performed for each of theobjective lenses to determine the distance between the marks on the ocular mi-crometer. The ocular micrometer can then be used to measure lengths andwidths of microbes and other objects on the specimen slide. The sizes of somemicroorganisms are shown in Table 2–1.

MICROSCOPES

The human eye, a telescope, a pair of binoculars, a magnifying glass, and a mi-croscope can all be thought of as various types of optical instruments. A micro-scope is an optical instrument that is used to observe tiny objects, often objectsthat cannot be seen at all with the unaided human eye. Each optical instrument

Microscopy 27

Organism(s) Dimension(s) Approximate Size (�m)

Viruses (most) Diameter 0.01–0.3

BacteriaCocci (spherical bacteria) Diameter average � 1Bacilli (rod-shaped bacteria) e.g., Escherichia coli (width � length) average � 1 � 3

Filaments (width) 1

FungiYeasts e.g., Candida albicans (diameter) 3–5Septate hyphae (hyphae with Width 2–15

cross-walls)Aseptate hyphae (hyphae without Width 10–30

cross-walls)

Pond water protozoaChlamydomonas Length 5–12Euglena Length 35–55Vorticella Length 50–145Paramecium Length 180–300Volvoxa Diameter 350–500Stentora Length (extended) 1000–2000

T A B L E 2 - 1 Relative Sizes of Microorganisms

aThese organisms are visible with the unaided human eye.

has a limit as to what can be seen using that instrument. This limit is referred toas the resolving power or resolution of the instrument. Resolving power is dis-cussed in more detail later. Table 2–2 contains the resolving powers for variousoptical instruments.

28 CHAPTER 2

Resolving Useful Type Power Magnification Characteristics

Brightfield 0.2000 �m �1000 Used to observe morphology of microorganismssuch as bacteria, protozoa, fungi, and algae in living (unstained) and nonliving (stained) stateCannot 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 seenUseful for examining spirochetesSlightly 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 organismPrimarily a diagnostic technique (immunofluorescence) to detect microorganisms in cells, tissue, and clinical specimensTraining required in specimen preparation and microscope operation

Transmission 0.0002 �m �200,000 Specimen can be viewed on screenelectron (0.2 nm) Excellent resolutionmicroscope Allows examination of cellular ultrastructure (TEM) and viruses

Specimen is nonlivingImage is two-dimensional

Scanning 0.0200 �m �10,000 Specimen can be viewed on screenelectron (20 nm) Three-dimensional view of specimenmicroscope Useful in examining surface structure of cells (SEM) and viruses

Specimen is nonlivingResolution is limited compared with TEM

T A B L E 2 - 2 Characteristics of Various Types of Microscopes

Simple Microscopes

A simple microscope is defined as a microscope containing only one magnifyinglens. Actually, a magnifying glass could be considered a simple microscope.Images seen when using a magnifying glass usually appear about 3 to 20 timeslarger than the object’s actual size. During the late 1600s, Anton vanLeeuwenhoek, who was discussed in Chapter 1, used simple microscopes to ob-serve many tiny objects, including bacteria and protozoa (Fig. 2–2). Because ofhis unique ability to grind glass lenses, scientists believe that Leeuwenhoek’ssimple microscopes had a maximum magnifying power of about �300 (300times).

Compound Microscopes

A compound microscope is a microscope that contains more than one magnify-ing lens. Although it is not known with certainty who the first person was to con-struct and use a compound microscope, Hans Jansen and his son Zacharias areoften 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 calledphotomicrographs.

Microscopy 29

Figure 2-2. (A) Leeuwenhoek’s microscopes were very simple devices. Each had a tiny glasslens, 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 wasabout 3 to 4 inches long. It was held very close to the eye. (B) Although his microscopes hada magnifying capability of only around �200 to �300, Leeuwenhoek was able to create re-markable 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.)

Because visible light (from a built-in light bulb) is used as the source of illu-mination, the compound microscope is also referred to as a compound light mi-croscope. It is the wavelength of visible light (approximately 0.45 �m) that lim-its 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 aresmaller than half of the wavelength of visible light. A compound light micro-scope is shown in Figure 2–4, and the functions of its various components are de-scribed in Table 2–3.

The compound light microscopes used in laboratories today contain twomagnifying lens systems. Within the eyepiece or ocular is a lens called the ocu-lar lens; it usually has a magnifying power of �10. The second magnifying lenssystem is in the objective, which is positioned immediately above the object tobe viewed. The four objectives used in most laboratory compound light micro-scopes are �4, �10, �40, and �100 objectives. As shown in Table 2–4, totalmagnification 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, spec-imens 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 canbe used to study algae, protozoa, and other large microorganisms. However, theoil-immersion objective (total magnification � �1000) must be used to studybacteria, because they are so tiny. To use the oil-immersion objective, a drop ofimmersion oil must first be placed between the specimen and the objective; theimmersion oil reduces the scattering of light and ensures that the light will enterthe oil immersion lens.

For optimal observation of the specimen, the light must be properly ad-justed and focused. The condenser, located beneath the stage, focuses light ontothe specimen, adjusts the amount of light, and shapes the cone of light enteringthe objective. Generally, the higher the magnification, the more light that isneeded.

Magnification alone is of little value unless the enlarged image possesses in-creased detail and clarity. Image clarity depends on the microscope’s resolving

30 CHAPTER 2

Hans Jansen, an optician in Middleburg, Holland, is often given credit for developingthe first compound microscope, sometime between 1590 and 1595. Although hisson, Zacharias, was only a young boy at the time, Zacharias apparently later tookover production of the Jansen microscopes. The Jansen microscopes contained twolenses and achieved magnifications of only �3 to �9. Compound microscopes hav-ing a three-lens system were used by Marcello Malpighi in Italy and Robert Hookein England, both of whom published papers between 1660 and 1665 describing theirmicroscopic findings. Some early compound microscopes are shown in Figure 2–3.

Early Compound Microscopes

Microscopy 31

Figure 2-3. A Leeuwenhoek microscope (center), surrounded by examples of early com-pound light microscopes. (Not to scale.)

power (or resolution), which is the ability of the lens system to distinguish be-tween 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 systemcan no longer resolve them as two separate objects (i.e., they are so close togetherthat they appear to be one object). That distance between them, where they ceaseto be seen as separate objects, is referred to as the resolving power of the opticalinstrument. Knowing the resolving power of an optical instrument also definesthe smallest object that can be seen with that instrument. For example, the re-solving power of the unaided human eye is approximately 0.2 mm. Thus, the un-aided human eye is unable to see objects smaller than 0.2 mm in diameter.

The resolving power of the compound light microscope is approximately1000 times better than the resolving power of the unaided human eye. In practi-cal terms, this means that objects can be examined with the compound micro-

Figure 2-4. A modern compound lightmicroscope.

32 CHAPTER 2

scope that are as much as 1000 times smaller than the smallest objects that canbe seen with the unaided human eye. Using a compound light microscope, wecan see objects down to about 0.2 �m in diameter.

Additional magnifying lenses could be added to the compound light micro-scope, but this would not increase the resolving power. As stated earlier, as long

Microscopy 33

Component Location Function

Ocular lens (also known as A �10 magnifying lensan eyepiece); a binocular microscope has two

Revolving nosepiece Above the stage Holds the objective lenses

Objective lenses Held in place above the stage Used to magnify objects placed by the revolving nosepiece on the stage

Stage Beneath the revolving nosepiece Flat surface upon which the specimen is placed

Stage adjustment knobs Beneath the stage Used to move the specimen(not shown in Fig. 2–2)

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 On the arm of the microscope, Used to focus the lensesknobs near the base

T a b l e 2 – 3 Components of the Compound Light Microscope

as visible light is used as the source of illumination, objects smaller than half ofthe wavelength of visible light cannot be seen. Increasing magnification withoutincreasing the resolving power is called empty magnification. It does no good toincrease 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 referredto as a brightfield microscope. If the regularly used condenser is replaced withwhat is known as a darkfield condenser, illuminated objects are seen against adark background (or “dark field”), and the microscope has been converted intoa darkfield microscope. In the clinical microbiology laboratory, darkfield mi-croscopy is routinely used to diagnose primary syphilis (the initial stage ofsyphilis). The etiologic (causative) agent of syphilis—a spiral-shaped bacterium,called Treponema pallidum—cannot be seen with a brightfield microscope be-cause it is thinner than 0.2 �m and, therefore, is beneath the resolving power ofthe compound light microscope. Treponema pallidum can be seen using a dark-field microscope, however, much in the same way that you can “see” dust parti-cles in a beam of sunlight. Dust particles are actually beneath the resolvingpower of the unaided eye and, therefore, cannot really be seen. What you see inthe beam is sunlight being reflected off the dust particles. With the darkfield mi-croscope, laboratory technologists do not really see the treponemes—they seethe light being reflected off the bacteria, and that light is easily seen against thedark background (Fig. 2–5).

Other types of compound microscopes include phase contrast microscopesand fluorescence microscopes. Phase contrast microscopes can be used to ob-serve unstained living microorganisms. Because the light refracted by living cellsis different from the light refracted by the surrounding medium, contrast is in-creased, and the organisms are more easily seen. Fluorescence microscopes con-tain a built-in ultraviolet (UV) light source. When UV light strikes certain dyesand pigments, these substances emit a longer wavelength light, causing them toglow against a dark background. Fluorescence microscopy is often used in im-munology laboratories to demonstrate that antibodies stained with a fluorescentdye have combined with specific antigens; this is a type of immunodiagnosticprocedure. (Immunodiagnostic procedures are described in Chapter 16.)

34 CHAPTER 2

Total Magnification Achieved When the Objective Objective Is Used in Conjunction With a �10 Ocular Lens

�4 (scanning objective) �40�10 (low-power objective) �100�40 (high-dry objective) �400�100 (oil immersion objective) �1000

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

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 wasdeveloped. It should be noted that electron microscopes cannot be used to ob-serve living organisms. Organisms are killed during the specimen processingprocedures. Even if they were not, they would be unable to survive in the vac-uum created within the electron microscope.

Electron microscopes use an electron beam as a source of illumination andmagnets to focus the beam. Because the wavelength of electrons traveling in avacuum is much shorter than the wavelength of visible light—about 100,000times shorter—electron microscopes have a much greater resolving power thancompound light microscopes. There are two types of electron microscopes:transmission electron microscopes and scanning electron microscopes.

A transmission electron microscope (Fig. 2–6) has a very tall column, at thetop of which an electron gun fires a beam of electrons downward. When an ex-tremely thin specimen (less than 1 �m thick) is placed into the electron beam,some of the electrons are transmitted through the specimen, and some areblocked. An image of the specimen is produced on a phosphor-coated screen atthe bottom of the microscope’s column. The object can be magnified up to ap-proximately 1 million times. Thus, using a transmission electron microscope, amagnification is achieved that is about 1000 times greater than the maximummagnification achieved using a compound light microscope. Even very tiny mi-crobes (e.g., viruses) can be observed using a transmission electron microscope.Because thin sections of cells are examined, transmission electron microscopyenables scientists to study the internal structure of cells. Special staining proce-dures are used to increase contrast between different parts of the cell. The firsttransmission electron microscopes were developed during the late 1920s and

Microscopy 35

Figure 2-5. Spiral-shapedTreponema pallidum, the etiologicagent of syphilis, as seen by dark-field microscopy. (Koneman EW,et al.: Color Atlas and Textbookof Diagnostic Microbiology, 5thed. Philadelphia, Lippincott-Raven, 1997.)

early 1930s, but it was not until the early 1950s that electron microscopes beganto be used routinely to study cells.

A scanning electron microscope (Fig. 2–7) has a shorter column and, insteadof being placed into the electron beam, the specimen is placed at the bottom ofthe column. Electrons that bounce off the surface of the specimen are capturedby detectors, and an image of the specimen appears on a monitor. Scanning elec-tron microscopes are used to observe the outer surfaces of specimens (i.e., sur-face detail). Although the resolving power of scanning electron microscopes(about 20 nm) is not quite as good as the resolving power of transmission elec-tron microscopes (about 0.2 nm), it is still possible to observe extremely tiny ob-jects using a scanning electron microscope. Scanning electron microscopes be-came available during the late 1960s.

Both types of electron microscopes have built-in camera systems. The pho-tographs taken using transmission and scanning electron microscopes are called

36 CHAPTER 2

Figure 2-6. Modern transmissionelectron microscopes are verysimilar in appearance to the onepictured here, being operated byone of the authors (P.G.E.) of thistextbook (c. 1980). The specimento be examined is placed into thecolumn at the point indicated bythe arrow. Note the porthole-type windows at the bottom ofthe column, through which an im-age of the specimen is viewed.The numerous knobs and dialscontrol the magnification, focus,and built-in camera system. Someof the transmission electron mi-crographs (TEMs) in this bookwere taken using the microscopepictured here.

Microscopy 37

transmission electron micrographs (TEMs) and scanning electron micrographs(SEMs), respectively. They are black and white images. If you ever see electronmicrographs in color, they have been artificially colorized. Figures 2–8, 2–9, and2–10 show the differences in magnification and detail between electron micro-graphs and light photomicrographs. Refer to Table 2–2 for the characteristics ofvarious types of microscopes.

Figure 2-7.Scanning electronmicroscope.

Figure 2-8. Staphylococcus aureus, as seen by light microscopy. (Original magnification,�1000.) (Photograph courtesy of W.L. Wong.)

38 CHAPTER 2

Figure 2-10. The three-dimensional qualities of scanningelectron microscopy clearly revealthe corkscrew shape of cells of thesyphilis-causing spirochete,Treponema pallidum, attached hereto rabbit testicular cells grown inculture. (Original magnification,�8000). (Volk WA, et al.:Essentials of Medical Microbiology,5th ed. Philadelphia, Lippincott-Raven, 1996.)

Figure 2-9. Staphylococcus aureus, as seen by transmission electron microscopy. (Originalmagnification, �40,000.) (Photograph courtesy of Ray Rupel.)

■ A meter (M) can be divided into 10 decime-ters, 100 centimeters, 1000 millimeters, 1 mil-lion micrometers, or 1 billion nanometers.

■ The metric system is used to describe thesizes of microorganisms. The sizes of bacte-ria and protozoa are expressed in microme-ters (�m), whereas the sizes of viruses areexpressed in nanometers (nm).

■ The development of simple and compoundlight microscopes enabled the discovery andvisualization of microorganisms. Simple mi-croscopes have only one magnifying lens,whereas compound microscopes have morethan one magnifying lens.

■ The limiting factor of compound light micro-scopes is the type of illumination being used.Because visible light is used as the source ofillumination, objects that are smaller thanhalf the wavelength of visible light cannot beseen. The resolving power (resolution) of thecompound light microscope is 0.2 �m.

■ Smaller objects can be seen using electronmicroscopes, because electrons are used asthe source of illumination. The wavelengthof electrons is shorter than that of visiblelight.

■ Transmission electron microscopes enablescientists to see inside of cells (i.e., to see in-ternal details). Using scanning electron mi-croscopes, scientists are able to study sur-face details. The resolving power of thetransmission electron microscope is 0.2 nm,whereas the resolving power of the scanningelectron microscope is 20 nm.

■ Because they are so tiny, most viruses canonly be seen using electron microscopes.

■ Photographs taken through the lens systemof the compound light microscope are calledphotomicrographs, whereas those takenwith electron microscopes are called trans-mission electron micrographs and scanningelectron micrographs.

Microscopy 39

REVIEW OF KEY POINTS

ON THE WEB—h t t p : / / c o n n e c t i o n . l w w . c o m / g o / b u r t o n 7 e

■ 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 howmany nanometers?

a. 100b. 1000c. 10,000d. 100,000e. 1,000,000

2. Assume that a pin head is 1 mmin diameter. How many spherical

bacteria (cocci), lined up side-by-side, would fit across the pinhead. (Hint: Use informationfrom Table 2–1.)

a. 10b. 100c. 1000d. 10,000e. 100,000

40 CHAPTER 2

3. What is the length of an averagerod-shaped bacterium (bacillus)?

a. 3 mmb. 3 �mc. 3 nmd. 0.3 mme. 0.03 mm

4. What is the total magnificationwhen using the high-power (high-dry) objective of a compoundlight microscope equipped with a�10 ocular lens?

a. �10b. �40c. �50d. �100 e. �400

5. How many times better is theresolution of the transmissionelectron microscope than the res-olution of the unaided humaneye?

a. 100b. 1000c. 10,000d. 100,000e. 1,000,000

6. How many times better is theresolution of the transmissionelectron microscope than the res-olution of the compound lightmicroscope?

a. 100b. 1000c. 10,000d. 100,000e. 1,000,000

7. How many times better is theresolution of the transmissionelectron microscope than the res-olution of the scanning electronmicroscope?

a. 100b. 1000c. 10,000d. 100,000e. 1,000,000

8. The limiting factor of any com-pound light microscope (i.e., thething that limits its resolution to0.2 �m) is the:

a. company from which it waspurchased.

b. number of condenser lensesit has.

c. number of magnifying lensesit has.

d. number of ocular lenses ithas.

e. wavelength of visible light.

9. Which of the following individu-als is given credit for developingthe first compound microscope?

a. Anton van Leeuwenhoekb. Hans Jansenc. Louis Pasteurd. Marcello Malpighie. Robert Hooke

10. A compound light microscopediffers from a simple microscopein that the compound light micro-scope contains more than one:

a. condenser lens.b. light bulb.c. magnifying lens.d. objective lens.e. ocular lens.