Prescott’s Microbiology, 9th Edition
2
Microscopy
CHAPTER OVERVIEW
This chapter provides a relatively detailed description of the bright-field microscope and its use. Other
common types of light microscopes are also described. Following this, various procedures for the preparation
and staining of specimens are introduced. The chapter continues with a description of the two major types of
electron microscopes and the procedures associated with their use. It concludes with descriptions of recent
advances in microscopy: electron cryotomography and scanning probe microscopy.
LEARNING OUTCOMES
After reading this chapter you should be able to:
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relate the refractive indices of glass and air to the path light takes when it passes through a prism or
convex lens
correlate lens strength and focal length
evaluate the parts of a light microscope in terms of their contributions to image production and use of the
microscope
predict the relative degree of resolution based on light wavelength and numerical aperture of the lens used
to examine a specimen
create a table that compares and contrasts the various types of light microscopes in terms of their uses,
how images are created, and the quality of images produced
recommend a fixation process to use when the microbe is a bacterium or archaeon and when the microbe
is a protist
plan a series of appropriate staining procedures to describe an unknown bacterium as fully as possible
compare what happens to Gram-positive and Gram-negative bacterial cells at each step of the Gramstaining procedure
create a concept map, illustration or table that compares transmission electron microscopes (TEM) to
light microscopes
decide when it would be best to examine a microbe by TEM, scanning electron microscopy (SEM), and
electron cryotomography
distinguish scanning tunneling from atomic force microscopes in terms of how they create images and
their uses
evaluate light microscopy, electron microscopy, and scanning probe microscopy in terms of their uses,
resolution and the quality of the image created
CHAPTER OUTLINE
I.
Lenses and the Bending of Light
A. Light is refracted (bent) when passing from one medium to another; the refractive index is a
measure of how greatly a substance slows the velocity of light; the direction and magnitude of
refraction is determined by the refractive indexes of the two media forming the interface
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Prescott’s Microbiology, 9th Edition
B.
Convex lenses bend parallel light rays from a distant light source and focus the light rays at a
specific place known as the focal point; the distance between the center of the lens and the focal
point is the focal length; convex lenses allow our eyes to focus at a much closer range
II. Light Microscopes
A. The bright-field microscope produces a dark image against a brighter background; the total
magnification of the image is the product of the magnification of the objective lens and the
magnification of the ocular (eyepiece) lens
B. Microscope Resolution
1. Microscope resolution refers to the ability of a lens to separate or distinguish between small
objects that are close together
2. The major factor determining resolution is the wavelength of light used; the
shorter the wavelength, the greater the resolution
3. The numerical aperture of the objective lens (ability to gather light) also
impacts resolution; the larger the numerical aperture, the greater the resolution
and the shorter the working distance of the lens
4. The unaided eye has a resolution of 0.2 mm; the typical light microscope has a
resolution 1,000 times greater
C. The dark-field microscope is used to observe living unstained preparations by creating a cone of
light such that only light reflected or refracted by the specimen is seen, thereby producing a bright
image of the object against a dark background
D. The phase-contrast microscope converts slight differences in refractive index and cell density into
easily detected variations in light intensity; it is an excellent way to observe inclusions in living cells
and studying microbial motility
E. The differential interference contrast (Nomarski) microscope detects differences in refractive
indices and thickness using two beams of polarized light, which form brightly colored, threedimensional images of living, unstained specimens
F. The fluorescence microscope excites a specimen with a single color of light and shows a bright
image of the object resulting from the fluorescent light emitted by the specimen; specimens are
usually stained with a fluorochrome; it is important for identification of pathogens with
fluorescently labeled antibodies and for localization of proteins tagged with green fluorescent
protein
G. Confocal microscopy
1. A focused laser beam is used to illuminate a point on a specimen (usually fluorescently
stained); light from the illuminated spot is focused by an objective lens; an aperture above the
lens blocks out stray light from parts of the specimen that lie above and below the plane of
focus; a detector measures the amount of illumination from each point, creating a digitized
signal
2. After examining many points (optical z-sections), a computer combines all the digitized
signals to form a three-dimensional image with excellent contrast and resolution, especially
valuable for examining living biofilms
III. Preparation and Staining of Specimens
A. Fixation refers to the process by which internal and external structures are preserved and fixed in
position; it usually kills the organism and firmly attaches it to the microscope slide
1. Heat fixing preserves overall morphology but not internal structures
2. Chemical fixing is used to protect fine cellular substructure and the morphology of larger,
more delicate microorganisms
B. Dyes and simple staining
1. Dyes have two common features: chromophore groups and the ability to bind cells by ionic,
covalent, or hydrophobic bonding
a. Basic dyes bind negatively charged molecules and cell structures
b. Acid dyes bind to positively charged molecules and cell structures
2.
Simple staining uses a single staining agent to stain a specimen
C. Differential staining is used to divide bacteria into separate groups based on their different reactions
to an identical staining procedure
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in any manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part.
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Prescott’s Microbiology, 9th Edition
1.
Gram staining is the most widely used differential staining procedure because it divides
bacterial species into two groups—gram positive and gram negative—based on cell wall
characteristics
a. The fixed smear is first stained with crystal violet, which stains all cells purple
b. Iodine is used as a mordant to increase the interaction between the cells and the dye
c. Ethanol or acetone is used to decolorize; this is the differential step because grampositive bacteria retain the crystal violet whereas gram-negative bacteria lose the crystal
violet and become colorless
d. Safranin is then added as a counterstain to turn the gram-negative bacteria pink while
leaving the gram-positive bacteria purple
2. Acid-fast staining is a differential staining procedure for cell walls that can be used to identify
two medically important species of bacteria—Mycobacterium tuberculosis, the causative agent
of tuberculosis, and Mycobacterium leprae, the causative agent of leprosy
D. Staining specific structures
1. Endospore staining is a double-stain technique by which bacterial endospores stain one color
and vegetative cells stain a different color
2. Negative staining is widely used to visualize diffuse capsules surrounding the bacteria; those
capsules are unstained by the procedure and appear colorless against a stained background
3. Flagella staining is a procedure in which mordants are applied with stains to increase the
thickness of flagella to make them easier to see
IV. Electron Microscopy
A. The transmission electron microscope (TEM)
1. The TEM has a resolution about 1,000 times better than that of the light microscope (0.5 nm
versus 0.2 µm) due to the short wavelength of the electron beam used to create the image
2. In TEM, electrons scatter when they pass through thin sections of a specimen; the transmitted
electrons (those that do not scatter) are used to produce an image of electron-dense objects on
a fluorescent screen
3. Specimen preparation for TEM involves procedures for cutting thin sections, chemical
fixation, drying, embedding in plastics, and staining with heavy atoms; other preparation
methods include negative staining, shadowing, and freeze-etching
B. The scanning electron microscope (SEM) uses electrons reflected from the surface of a specimen to
produce a three-dimensional image of its surface features; many SEMs have a resolution of 7 nm or
less; specimen preparation usually involves chemical fixation, drying, and coating with metals
C. Electron cryotomography uses samples rapidly frozen to extremely low temperatures preserving
internal features and an imaging method where the sample is viewed form many angles to create
three-dimensional images
V. Scanning Probe Microscopy
A. Scanning probe microscopy measures surface features by moving a short probe over the object’s
surface
B. The scanning tunneling microscope creates an image using a probe that is one atom thick at its tip;
as it moves horizontally over the surface, tunneling currents change with distance from the
specimen; the vertical motion of the probe is used to create a three-dimensional image of the
specimen’s surface atoms; resolution is such that individual atoms can be observed
C. The atomic force microscope uses a very small amount of force on the probe tip to maintain a
constant distance between the tip and the specimen; the vertical movement of the probe across the
surface of the specimen is used to create a three-dimensional image; because it does not make use of
a tunneling current, it is useful for surfaces that do not conduct electricity well
CRITICAL THINKING
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© 2014 by McGraw-Hill Education. This is proprietary material solely for authorized instructor use. Not authorized for sale or distribution
in any manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part.

Prescott’s Microbiology, 9th Edition
1.
Explain why it is possible to increase the magnification of the light microscope above 1,500 and yet not
be able to see any additional details.
2.
Compare and contrast electron microscopy with light microscopy. Include in your answer the operation of
the instruments, the degree of magnification and resolution possible, and the procedures used for
preparation, fixation, and staining of specimens. What major advances in our knowledge of cell structure
were made possible by the invention of the electron microscope that were not possible with the light
microscope?
3.
Explain why it is necessary to use immersion oil to enhance the resolution of many light microscopes
when visualizing bacteria.
4
© 2014 by McGraw-Hill Education. This is proprietary material solely for authorized instructor use. Not authorized for sale or distribution
in any manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part.
