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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.
CHAPTER OBJECTIVES
After reading this chapter you should be able to:
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•
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describe how lenses bend light rays to produce enlarged images of small objects
describe the various parts of the light microscope and how each part contributes to the functioning of the
microscope
describe the preparation and simple staining of specimens for observation with the light microscope
describe the Gram-staining procedure and how it is used to categorize bacteria
describe the basis for the various staining procedures used to visualize specific structures associated with
microorganisms
compare the operation of the transmission and scanning electron microscopes with each other and with
light microscopes
describe dark-field microscopy, phase-contrast microscopy, differential interference contrast microscopy,
confocal microscopy, electron cryotomography, and scanning probe microscopy
compare and contrast light microscopes, electron microscopes, confocal microscopes, and scanning probe
microscopes in terms of their resolution, the types of specimens that can be examined, and the images
produced
CHAPTER OUTLINE
I.
II.
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
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
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
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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
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
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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
TERMS AND DEFINITIONS
Place the letter of each term in the space next to the definition or description that best matches it.
____ 1.
____ 2.
____ 3.
____ 4.
____ 5.
____ 6.
The bending of light rays at the interface of one
medium with another
A measure of how greatly a substance changes the
velocity of light, and a factor determining the direction
and magnitude of the bending of light rays
The point at which a lens focuses rays of light
The distance between the center of a lens and the focal
point
Conventional microscope that produces a dark image
against a brighter background
Describes a microscope whose image remains in focus
when the objectives are changed
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____ 7.
____ 8.
____ 9.
The ability of a lens to
separate or distinguish
small objects that are
close together
A microscope that uses
two beams of polarized
light to form threedimensional images of
living, unstained
specimens
The distance between
the front surface of the
____ 10.
____ 11.
____ 12.
____ 13.
____ 14.
____ 15.
____ 16.
____ 17.
____ 18.
____ 19.
lens and the surface of the cover glass (or the
specimen) when the specimen is in sharp focus
A microscope that produces a bright image of the
specimen against a dark background
A microscope that converts slight differences in
refractive index and cell density into easily detected
variations in light intensity
A microscope that exposes specimens to ultraviolet,
violet, or blue light and forms an image from the
resulting light emitted, which has a different
wavelength
The process by which the internal and external
structures of cells and organisms are preserved and
maintained in position
A staining process in which a single staining agent is
used
A staining process that divides organisms into two or
more separate groups depending on their reaction to
the same staining procedure
A substance that accelerates the reaction of cell
structures with a dye so that the cell is more intensely
stained
A staining procedure in which the background is dark
and the organism remains unstained
A staining process in which heat is used to increase the
affinity of bacterial endospores to dye; endospores are
usually resistant to simple staining procedures
A staining process that enables the observation of thin,
threadlike flagella by increasing their thickness and
then staining them
____ 20. A microscope that forms an image by
focusing a beam of electrons on a
specimen
____ 21. An electron microscope that creates an
image from transmitted electrons (those
not scattered when they pass through a
thin section of a specimen)
____ 22. A staining process in which heavy
metals are applied to specimens at an
approximately 45 angle; this provides
a three-dimensional image similar to
shadowing with light
____ 23. An electron microscope that creates an
image from electrons emitted from the
surface of a specimen that has been
excited by a beam of focused electrons
a.
b.
c.
d.
e.
f.
g.
h.
i.
j.
k.
l.
m.
n.
o.
p.
q.
r.
s.
t.
u.
v.
w.
x.
y.
z.
atomic force microscope
bright-field microscope
dark-field microscope
differential interference
contrast (DIC) microscope
differential staining
electron microscope
fixation
flagella staining
fluorescence microscope
focal length
focal point
freeze-etching
mordant
negative staining
parfocal
phase-contrast microscope
refraction
refractive index
resolution
scanning electron microscope
(SEM)
scanning tunneling electron
microscope
shadowing
simple staining
spore staining
transmission electron
microscope (TEM)
working distance
____ 24. A procedure in which frozen specimens
are broken along lines of greatest
weakness, usually down the middle of
internal membranes; exposed surfaces
are then shadowed for production of a
better image
____ 25. A scanning probe microscope that uses
voltage flow between the tip of the
probe and the electron clouds of the
surface atoms of the specimen
____ 26. A scanning probe microscope that is
useful for surfaces that do not conduct
electricity well
MICROSCOPE IDENTIFICATION
Using the terms listed below, label the parts of the microscope indicated on the accompanying picture. In the
space beside each term provide a brief description of its function.
1.
Ocular (eyepiece) lens:
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2.
Arm:
3.
Objective lens:
4.
Coarse focus adjustment knob:
5.
Fine focus adjustment knob:
6.
Base with light source:
7.
Nosepiece:
8.
Mechanical stage:
9.
Substage condenser:
10. Aperture diaphragm control:
11. Stage adjustment knobs:
12. Light intensity control:
13. Body assembly:
14. Field diaphragm lever:
13
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GRAM-STAINING PROCEDURE
Complete the table for the Gram-staining procedure by supplying the missing information.
Color after Completion of Step
Procedure Step
Reagent
Gram Positive
Gram Negative
____________
____________
____________
Iodine
____________
Purple
3. Decolorizer
____________
____________
Colorless
4. Counterstain
____________
____________
____________
1. Primary stain
2. ____________
LENSES AND MAGNIFICATION
Complete the table below by filling in the missing information.
Ocular Lens
Objective Lens
Magnification
1.
10
40
________
2.
10
________
1000
3.
15
________
600
FILL IN THE BLANK
1.
2.
3.
4.
5.
6.
7.
The objective lens forms an enlarged image within the microscope called the ____________ image. The
eyepiece lens further magnifies this image to form the ____________ image, which appears to lie just
beyond the stage about 25 cm away.
Thin films of bacteria that have been air-dried onto a glass microscope slide are called ____________.
A
microscope uses lenses made of glass to focus light onto a specimen, while an electron
microscope uses magnetic lenses to focus beams of
onto a specimen.
Special dyes called ____________ are used in fluorescence microscopy. These dyes are excited by light
with a specific wavelength and emit light with a ____________ wavelength, thus having less energy than
the light originally absorbed. In this way the dye gives up its trapped energy and returns to a more stable
state.
The presence of diffuse capsules surrounding many bacteria is commonly revealed by ____________
staining, in which the background is stained dark. The cell can also be counterstained for greater
visibility, leaving the capsule colorless.
Although there are many types of dyes, all share two common features:
groups,
which give dyes their colors, and the ability to bind to cells by ionic, covalent, and hydrophobic bonds.
Those that bind by ionic bonds are called ionizable dyes. They can be divided into two broad classes
based on the nature of their charged group. Dyes such as methylene blue and crystal violet have positively
charged groups and are called
dyes. Dyes such as eosin have negatively charged groups and
are called
dyes.
Electron cryotomography uses specimens that are rapidly
to preserve internal structures and
produces three-dimensional images by recording images from many directions in what is called a
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.
MULTIPLE CHOICE
For each of the questions below select the one best answer.
1.
2.
3.
4.
5.
6.
Acid-fast organisms such as
Mycobacterium tuberculosis resist
decolorization by acid-alcohol solutions
because of the high concentration of
____________ in their cell walls.
a. proteins
b. carbohydrates
c. lipids
d. peptidoglycan
Why are smears heat-fixed prior to
staining?
a. to kill the organism
b. to preserve the internal structures
c. to attach the organism firmly to the
slide
d. All of the above are correct.
Which type of microscope is best for
visualizing small morphological features
within the cell interior?
a. light microscope
b. transmission electron microscope
c. dark-field microscope
d. scanning electron microscope
Transmission electron microscopy
requires the use of thin slices of a
microbial specimen. What should the
thickness of the specimen be?
a. 20 to 100 mm
b. 100 to 200 nm
c. 20 to 100 nm
d. 0.2 to 10 nm
For transmission electron microscopy, a
specimen can be spread out in a thin film
with uranyl acetate, which does not
penetrate the specimen. What is this
procedure called?
a. negative staining
b. shadowing
c. freeze-etching
d. simple staining
Which type of microscope best reveals
surface features of an organism?
a. fluorescence microscopy
b. phase-contrast microscopy
c. scanning electron microscopy
d. transmission electron microscopy
7.
As the magnification of a series of objective
lenses increases, what happens to the working
distance?
a. It increases.
b. It decreases.
c. It stays the same.
d. It cannot be predicted.
8. The Gram-staining procedure differentiates
bacteria based on the chemical composition of
which cell structure?
a. cytoplasmic membrane
b. cell wall
c. cytoplasm
d. chromosome
9. What is the distance between the focal point of a
lens and the center of the lens called?
a. working distance
b. numerical aperture
c. focal length
d. parallax distance
10. A microscope is able to keep objects in focus
when the objective lens is changed. What term
is used to describe this property?
a. equifocal
b. parfocal
c. optically constant
d. focally constant
11. Which microscope is especially useful for
examining thick specimens such as biofilms?
a. transmission electron microscope
b. dark-phase-contrast microscope
c. confocal scanning laser microscope
d. bright-field light microscope
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TRUE/FALSE
____ 1.
Light is refracted at the interface between two materials with different refractive indexes because the
velocity of light is altered.
____ 2. Resolution becomes greater as the wavelength of the illuminating light decreases.
____ 3. Phase-contrast microscopy enhances density differences among internal cellular structures and
therefore allows these structures to be visualized without stains or dyes.
____ 4. Basic dyes are cationic (positively charged) and are commonly used to stain bacteria since the
surfaces of these organisms are usually negatively charged.
____ 5. The Gram-staining procedure is one of the most widely used differential stains because it divides
bacterial species into two groups: gram-positive and gram-negative.
____ 6. Since transmission electron microscopy uses electrons rather than light, it is not necessary to stain
biological specimens before observation.
____ 7. Freeze-etching minimizes the production of artifacts since the cells are not subjected to chemical
fixation, dehydration, or plastic embedding.
____ 8. The resolution of a microscope is not related to its magnification. Therefore, although it is possible
to build a light microscope capable of 10,000 magnification, it would only be magnifying a blur.
____ 9. Scanning tunneling electron microscopes can be used to visualize individual atoms.
____ 10. Denser regions of a specimen scatter more electrons and therefore appear darker in the image
projected onto the screen of a transmission electron microscope.
11. The larger the numerical aperture of the objective lens, the greater the resolution of the microscope.
CRITICAL THINKING
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?
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ANSWER KEY
Terms and Definitions
1. q, 2. r, 3. k, 4. j, 5. b, 6. o, 7. s, 8. d, 9. z, 10. c, 11. p, 12. i, 13. g, 14. w, 15. e, 16. m, 17. n, 18. x,
19. h, 20. f, 21. y, 22. v, 23. t, 24. l, 25. u, 26. a
Gram-Staining Procedure
1. crystal violet; purple; purple 2. mordant; purple 3. ethanol or acetone; purple 4. safranin; purple; red
Lenses and Magnification
1. 4002. 100, 3. 40
Fill in the Blank
1. real; virtual 2. smears 3. light; electrons 4. fluorochromes; longer 5. negative 6. chromophore; basic; acid 7.
frozen; tilt series
Multiple Choice
1. c, 2. d, 3. b, 4. c, 5. a, 6. c, 7. b, 8. b, 9. c, 10. b 11. c
True/False
1. T, 2. T, 3. T, 4. T, 5. T, 6. F, 7. T, 8. T, 9. T, 10. T, 11. T
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