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Chapter 3
Observing Microorganisms
Through a Microscope
Units of Measurement
Microorganisms are measured by metric units unfamiliar to many of us. The micrometer (m), formerly
known as the micron, is equal to 0.000001 (10–6) meter. The prefix micro indicates that the unit following
should be divided by one million. A nanometer (nm), formerly known as a millimicron (m) is equal
to 0.000000001 (10–9) meter. Nano tells us that the unit should be divided by one billion. An angstrom
(Å) is equal to 0.0000000001 (10–10) meter.
Microscopy: The Instruments
Compound Light Microscopy
The compound light microscope has two sets of lenses: the objective and the ocular. Specimens magnified by the objective lens—magnified 100 times, for example—are remagnified by the ocular, usually
10 times. Thus, the total magnification is 1000 times. Most microscopes provide magnifications of 100, 400,
and 1000. A magnification of 2000 times is about the highest obtainable. The specimen is illuminated by
visible light from the light source—the illuminator—that is passed through a condenser, which directs the
light rays through the specimen (Figure 3.1a). Resolution, or resolving power, is the ability of a microscope to distinguish between two points. The shorter the wavelength of the illumination, the better the
resolution. The white light used in a compound light microscope limits resolving power to about 0.2 m.
The lenses closest to the specimen are the objective lenses. For the highest magnification, it is necessary to use oil immersion objectives. Immersion oil has the same refractive index as glass; that is,
the relative velocity of light passing through it is the same. Without immersion oil filling the space
between the slide bearing the specimen and the objective, the image will be fuzzy, with poor resolution
(Figure 3.1b).
Darkfield Microscopy
Some microorganisms, such as the thin spirochete Treponema pallidum, which causes syphilis, are best
seen with darkfield microscopy. In the darkfield microscope, an opaque disk blocks light from entering
the objective directly. The light hits only the sides of the specimen, and scattered light enters the objective and reaches the eyes. The specimen appears white against a black background.
Phase-Contrast Microscopy
Living microorganisms do not show up well in the ordinary compound light microscope. The phasecontrast microscope takes advantage of subtle differences in the refractive index of different parts of
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Chapter 3
Ocular lens
(eyepiece)
Remagnifies the
image formed by
the objective lens
Ocular lens
Line of vision
Path of light
Body tube Transmits
the image from the objective lens to the ocular lens
Prism
Body tube
Arm
Objective lenses Primary
lenses that magnify the
specimen
Objective
lenses
Stage Holds the
microscope slide
in position
Specimen
Condenser Focuses
light through specimen
Condenser
lenses
Diaphragm Controls the amount
of light entering the condenser
Illuminator
Coarse focusing knob
Base with
source of
illumination
Illuminator Light source
Base
Fine focusing knob
FIGURE 3.1
(a) Principal parts and functions
(b) The path of light (bottom to top)
The compound light microscope.
the living cell and its surrounding medium. As light is slowed down in portions of differing density, it
travels slightly different pathways. When recombined for viewing, the “phase differences” are seen as
areas of differing brightness. The microorganism (and many of its internal structures) is seen in its natural state, alive and unstained.
Differential Interference Contrast (DIC) Microscopy
Differential interference contrast microscopy is similar to phase-contrast microscopy. It uses differences in refractive indexes but uses two beams of light instead of one. Prisms split each light beam,
adding contrasting colors. Compared to standard phase-contrast microscopes, DIC images are more
brightly colored, are nearly three-dimensional in appearance, and have higher resolution.
Fluorescence Microscopy
Certain fluorochrome dyes, which glow with visible light—yellow, for example—when illuminated by
ultraviolet light, can be used to view and identify microorganisms. This is fluorescence; certain substances, when illuminated by a short wavelength, emit light of a longer wavelength. Fluorescence
microscopy techniques use a special microscope with ultraviolet light illumination; this light illuminates
the specimen but is not permitted to reach the eye. The stained microorganism is highly visible against
a dark background in such a microscope. However, the principal use of these dyes and microscopes is
in the fluorescent-antibody technique, or immunofluorescence. In this technique, the organisms are
allowed to react on a slide with antibodies (highly specific proteins produced by the body’s defense
Observing Microorganisms Through a Microscope
Antibodies
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Fluorochrome
Antibodies
combined with
fluorochrome
Unknown bacterium
Cell-surface
antigen molecules
Bacterial cell with bound antibodies
combined with fluorochrome
FIGURE 3.2 The principle of immunofluorescence. A type of fluorochrome is combined with antibodies against a
specific type of bacterium. When the preparation is added to bacterial cells on a microscope slide, the antibodies
attach to the bacterial cells and the cells fluoresce when illuminated with ultraviolet light.
system). A fluorescent dye is attached to the antibody. The combination of the antibody, the attached
dye, and the microorganism for which the antibody is specific (called an antigen; it stimulates the body
to produce these antibodies) allows the microorganism’s presence to be detected (Figure 3.2). Because
the antibody is specific for a particular microorganism, this is a very useful diagnostic technique. It is
often used for diagnosis of syphilis and rabies.
Confocal Microscopy
One plane of part of a specimen in confocal microscopy is illuminated with a laser, which passes the
returned light through an aperture aligned with the illuminated region. Successive planes and regions
can be scanned and a clear two-dimensional image obtained. When used with computers, these images
can be used to construct three-dimensional images.
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Chapter 3
Electron Microscopy
The wavelengths of electrons, which travel in waves much as light does, are only about 1/100,000 as
long as those of visible light and therefore have much better resolving power. They can be focused by
magnetic lenses in an electron microscope.
Transmission Electron Microscopy. In transmission electron microscopy, a beam of electrons is
passed through ultrathin sections of the specimen and focused on a fluorescent screen, where it is visible
to the eye and can be photographed. Objects are generally magnified 10,000 times to 100,000 times, and
structures, called artifacts, may appear as a result of the method of preparation.
Salts of heavy metals may be fixed to the specimen (positive staining) to increase the density and
make a darker image. If the metals are used on the surrounding field, it is termed negative staining,
which is useful for viewing exceptionally small specimens. The technique of shadow casting produces a
three-dimensional effect by spraying a heavy metal at an angle, accumulating on one side and leaving a
clear area on the other. This also provides an idea of the size and shape.
Scanning Electron Microscope. In scanning electron microscopy, the electron beam is directed at
the intact specimen from the top, rather than passing through a section, and electrons leaving the surface
of the specimen (secondary electrons) are viewed on a televisionlike screen. Spectacular pictures of seemingly three-dimensional, intact organisms are possible. Objects are generally magnified 1000 times to
10,000 times with a resolving power of about 20 nm.
Scanning Tunneling and Atomic Force Microscopy
Scanning tunneling microscopy uses a thin metal probe that scans a specimen and produces an image
of the bumps and depressions of the atoms on the surface of the specimen. In atomic force microscopy
a metal-and-diamond probe is moved along the surface of the specimen. The recorded movements yield
a three-dimensional image.
Preparation of Specimens for Light Microscopy
Preparing Smears and Staining
Most microorganisms are viewed in stained preparations; that is, they are colored with a dye to make
them visible or to emphasize certain structures. A thin film of a microbial suspension, called a smear, is
spread on the surface of a slide. Flaming the air-dried smear coagulates the microbial proteins and fixes
the microorganisms to the slide so they do not wash off. The smear can then be stained. Basic dyes
have a colored ion that is positive, helping them adhere to bacteria, which are slightly negative.
Examples of basic dyes are crystal violet, methylene blue, and safranin. Acidic dyes, having a negative
color ion, are more attracted to the background than to the negatively charged bacteria; thus, a field of
colorless bacteria is presented against a stained background. This is an example of negative staining.
An example of an acidic dye is eosin.
Simple Stains
To visualize shapes and arrangements of cells, a simple stain is usually sufficient. A chemical called a
mordant may be added to make the microorganism stain more intensely or increase its size to enhance
visibility.
Differential Stains
The most useful differential stain is the Gram stain, developed by Hans Christian Gram. It divides
bacteria into two large groups: gram-positive and gram-negative. In preparing a Gram stain (1) apply a
Observing Microorganisms Through a Microscope
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purple dye, crystal violet, to a heat-fixed smear. This stains all the cells and is called the primary stain.
After a water rinse, (2) an iodine mordant is added. When a smear stained in this manner is (3) washed
with ethanol or an ethanol-acetone solution, some species of bacteria are decolorized and others are not.
If the smear retains the purple dye, the organism is gram-positive. If the alcohol removes the dye, the
colorless microorganisms are no longer visible. (4) Safranin, a red dye, is then applied and the decolorized, or gram-negative, bacteria appear pink. Safranin is used here as a counterstain. The Gram stain
reflects a basic difference in the cell wall structure of bacteria. It is a first step in identification, and the
susceptibility of microorganisms to antibiotics is often related to the Gram reaction.
Acid-Fast Stain. Members of the genera Mycobacterium (which includes the causes of tuberculosis
and leprosy) and Nocardia possess a cell wall with waxy components. The red dye carbolfuchsin is more
soluble in these waxes than in acid-alcohol and is retained by the cell. Therefore, the acid-fast stain, in
which carbolfuchsin is applied and gently steamed for several minutes, will stain them red. This dye is
held so firmly that the cells are not decolorized by acid-alcohol, which does remove the dye from bacteria that are not acid-fast. A methylene blue counterstain will produce a slide in which acid-fast organisms are red and others are blue. The acid-fast stain is an invaluable aid in the diagnosis of tuberculosis
and leprosy.
Special Stains
A colloidal suspension of dark particles such as India ink or nigrosin can be used as a capsule stain.
The capsule will appear around each bacterial cell as a halo from which the India ink carbon particles
are excluded. Endospores do not stain by ordinary methods, but the Schaeffer–Fulton endospore stain,
which uses malachite green as a primary stain and safranin as a counterstain, shows endospores as
green within red or pink cells. Flagella are too small to be resolved by light microscopes. In a flagella
stain, a mordant can be used to increase the diameter of the flagella until they are visible in a light
microscope.
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Chapter 3
Self-Tests
In the matching section, there is only one answer to each question; however, the lettered
options (a, b, c, etc.) may be used more than once or not at all.
I. Matching
1. The electrons pass through a thin section of the specimen.
a. Compound light microscope
2. Visible light passes through the specimen; uses separate
objective and ocular lenses.
b. Scanning electron microscope
3. Details become visible because of differences in the refractive index of different parts of the cell.
4. Visible light is scattered after striking the specimen, and
the specimen is visible against a darkened background.
5. A special microscope using ultraviolet illumination.
c. Phase-contrast microscope
d. Transmission electron
microscope
e. Fluorescence microscope
f. Darkfield microscope
6. The electrons strike the surface of the specimen, and secondary electrons leaving the surface are viewed on a
televisionlike screen.
II. Matching
1. Pertaining to the relative velocities of light through a
substance.
2. Involves the use of antibodies and ultraviolet light.
3. One millionth of a meter.
4. One ten-billionth of a meter.
5. The ability to separate two points in a microscope field.
a. Micrometer
b. Nanometer
c. Ångstrom
d. Resolving power
e. Refractive index
f. Immersion oil
g. Immunofluorescence
Observing Microorganisms Through a Microscope
III. Matching
1. Adhere(s) best to bacteria, which have a negative charge,
because the color molecule has a positive charge.
2. Used in diagnosis of tuberculosis.
3. Involve(s) the use of a negative stain made from India ink
particles.
4. Schaeffer–Fulton stain.
5. Use(s) carbolfuchsin dye.
a. Basic dyes
b. Acidic dyes
c. Gram stain
d. Acid-fast stain
e. Capsule stain
f. Endospore stain
6. Use(s) malachite green.
7. Reflect(s) a basic difference between microbial cell walls;
ethanol will not remove stain from bacteria.
IV. Matching
1. A microscope that uses laser illumination.
a. Confocal
2. Extremely thin microbes, for example, the spirochete
Treponema pallidum, are best seen with this type of light
microscope.
b. Phase contrast
3. This type of electron microscope yields images with seemingly three-dimensional views of the specimen.
c. Darkfield
d. Transmission
e. Scanning
4. Light rays that pass through different portions of the specimen reach the eye with their wave-peaks reinforced or cancelled, making structures of the specimen relatively light
or dark.
V. Matching
1. Formerly known as a micron.
a. Micrometer
2. Formerly known as a millimicron.
b. Nanometer
3. This is 10–10 of a meter.
c. Ångstrom
4. A billionth of a meter.
d. Millimeter
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Chapter 3
Fill in the Blanks
1. About the highest magnification possible in a compound light microscope is
.
2. Immersion oil has about the same refractive index as
.
3. Fluorochrome dyes glow with visible light when illuminated by
light.
4. Electron wavelengths are only about 1/100,000 as long as visible light and therefore have much
resolving power. (better, poorer)
5. Bacteria tend to have a slightly
electrical charge. (positive, negative)
6. The thin film of a microbial suspension spread on the surface of a slide is called a
.
7. Flaming the slide before applying the stain is called
.
8. Transmission electron microscopy permits magnifications as high as about 10,000 times to
.
9. In the flagella stain, a
is used to increase the diameter of the flagella.
10. Two bacterial genera that are acid-fast are
and
.
11. A disease for which the acid-fast stain is useful in diagnosis is
12. In order to see shapes and arrangements of cells, a
sufficient.
13.
dyes have a negative color ion. (acidic, basic)
.
stain is usually
Observing Microorganisms Through a Microscope
33
Label the Art
a.__________________
Line of vision
Path of light
Prism
Body tube
b.__________________
Specimen
c.__________________
Illuminator
Base with
source of
illumination
Critical Thinking
1. The equation that describes the resolving power of a microscope is:
Resolving power = Wavelength of illumination/2 Numerical Aperture
(The numerical aperture of an oil immersion objective is usually 1.30.)
If the wavelength of light is 0.52 µm, what is the resolving power of this objective?
2. What type of microscopy would be most appropriate for viewing the following specimens or for the
following situations?
a. To identify pathogenic bacteria in clinical specimens.
b. To view objects smaller than 0.2 m, such as viruses.
c. To view heat-fixed, stained bacterial cells.
d. To view microorganisms that can’t be stained by standard methods, such as Treponema pallidum.
e. To view the internal structure of living microorganisms.
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Chapter 3
3. For each of the following specimens or situations, indicate which stain(s) or technique would be
most appropriate.
a. To detect bacterial capsules and evaluate an organism’s virulence.
b. To provide the necessary contrast for viewing specimens with a compound light microscope.
c. To diagnose infections of Mycobacterium or Nocardia.
d. To help determine what antibiotic will be most effective against a certain disease organism.
4. Why do gram-positive cells retain the crystal violet through the alcohol wash of Gram staining,
whereas gram-negative cells do not?
Answers
Matching
I.
II.
III.
IV.
V.
1. d
1. e
1. a
1. a
1. a
2. a
2. g
2. d
2. c
2. b
3. c
3. a
3. e
3. e
3. c
4. f
4. c
4. f
4. b
4. b
5. e 6. b
5. d
5. d 6. f 7. c
Fill in the Blanks
1. 2000 2. glass 3. ultraviolet 4. better 5. negative 6. smear 7. fixing 8. 100,000
9. mordant 10. Mycobacterium; Nocardia 11. tuberculosis or leprosy 12. simple 13. acidic
Label the Art
a. ocular lens
b. objective lenses
c. condenser lenses
Critical Thinking
1. It would be about 0.2 µm. (The figure for the wavelength of light is for green light, changed from
520 nm.)
2. a. Fluorescence microscopy
b. Electron microscopy
c. Brightfield microscopy
d. Darkfield microscopy
e. Phase-contrast microscopy
Observing Microorganisms Through a Microscope
35
3. a. A negative stain using India ink or nigrosin. The India ink (or nigrosin) stains the background
but doesn’t penetrate the capsule. The capsule shows up as a halo surrounding the cell against a
dark background.
b. For this purpose a simple stain such as safranin or methylene blue will work fine.
c. Acid-fast staining would be appropriate to diagnose infections of Mycobacterium and Nocardia.
The red dye, carbolfuchsin, binds strongly to a waxy substance in the cell wall of these organisms
but not to other “nonacid-fast” bacteria.
d. The Gram staining reaction is helpful information when choosing an antibiotic, which often
shows specificity for either gram-positive or gram-negative bacteria.
4. When iodine is added to a smear after previous staining with crystal violet, they combine to form a
complex (CV-I complex) that is larger than the crystal violet molecule that initially entered the cells.
The CV-I complex is too large to be washed out of the intact peptidoglycan layer of gram-positive
cells. When decolorizing gram-negative cells, the alcohol washes away the outer lipoprotein layer
and the crystal violet from the thin layer of peptidoglycan.