CHAPTER 3
MICROSCOPY AND STAINING
 Chapter Overview
Microbiology as a science could not begin until the development of a microscope powerful
enough to observe microorganisms. Furthermore, it required an inquisitive mind to observe these
"animalcules" and record their appearance and behavior. Once they were discovered, it still required
many years for the connection between microbes and disease to be made. The beginning of the
process was the development of the microscope.
The ability to magnify and visualize any object is based on many fundamental principles of
light. These principles are often overlooked even though an understanding of these principles is
essential to an understanding of some of the more complex microscopic techniques, such as phasecontrast microscopy, dark-field microscopy, and the electron microscope.
Following the presentation of the principles of microscopy, a survey of the most commonly
used light microscopes in microbiology is provided. This survey is followed by a thorough review
of electron microscopes, whose advent helped open and/or improve such fields as virology,
immunology, histology, and cell biology.
The last section is devoted to a presentation of the principles of some of the most important
stains used in microbiology, including the Gram, acid-fast, and endospore stains. Much of this last
material can and should be reemphasized and demonstrated in the laboratory.
 Chapter Objectives
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Describe briefly the evolution of microscopic instruments and their relationship to the progress
made in microbiology.
List the appropriate metric units that are used to measure bacteria, viruses, fungi, protozoa, and
helminths.
Contrast the differences and relationships among the following: wavelength, resolution, and
numerical aperture.
Discuss the relationship of microbiology to the following properties of light: transmission,
absorption, fluorescence, luminescence, phosphorescence, reflection, refraction, and diffraction.
Locate and describe the function of each major part of a compound light microscope.
List and describe the special adaptations of bright-field, dark-field, phase-contrast, differential
interference contrast, and fluorescence (UV) microscopy.
Briefly explain the principles of transmission, scanning tunneling, and scanning electron
microscopy, noting especially how each differs from light microscopy.
Compare the advantages and limitations of electron microscopy with those of light microscopy.
List and describe the techniques used to prepare and heighten contrast in specimens to be viewed
with a light microscope.
Define the term stain; list and describe the features and purpose of at least four common types of
stains used in microbiology.
List the steps in the Gram stain and explain each of their functions and results.
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 Web Destinations
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http://www.infochembio.ethz.ch/links/en/mikrobio_gramfaerbung.html
Microbiology staining techniques
http://www.microscopyu.com/
Nikon microscopy web site
http://www.ruf.rice.edu/~bioslabs/methods/microscopy/microscopy.html
Microscopy tutorial
http://www.bact.wisc.edu/Microtextbook/TOC.html
On-line microbiology textbook
http://sciencespot.net/Pages/classbio.html#micro
The Microscope Mania website is directed towards younger learners but still has good
information about microscopes and how they can be used.
http://www.microscopy.fsu.edu/primer/java/scienceopticsu/powersof10/index.html
Secret Worlds: The Universe Within is an interesting website that starts out very large (the
universe) and goes very small (atoms) by powers of 10.
http://www.microscopy.fsu.edu/optics/olympusmicd/index.html
Olympic digital microscope information, tutorials, and image gallery.
 Discussion Topics
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Some bacteria found deep in the ocean are phosphorescent. What value does there seem to be for
these microbes, since there is no light down that deep?
Why are heavy metals such as gold used in staining specimens for electron microscope work?
 Track It Down
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What wavelengths of visible light are used in microscopy? How are those wavelengths selected?
Can viruses be identified with fluorescent dyes? Which viruses are identified and how do the
dyes work?
 Chapter Outline
I.
Historical Microscopy
A.
Early history
B.
Anton van Leeuwenhoek
II.
Principles of Microscopy
A.
Metric units
1.
Micrometer
3-2
B.
C.
III.
2.
Nanometer
3.
Angstrom
Properties of light: wavelength and resolution
1.
Wavelength
2.
Electromagnetic spectrum
3.
Resolution
a.
Factors affecting resolution
b.
Resolving power
c.
Numerical aperture
Properties of light: light and objects
1.
Reflection
2.
Transmission
3.
Absorption
a.
Luminescence
b.
Fluorescence
c.
Phosphorescence
5.
Refraction
a.
Index of refraction
b.
Effect of immersion oil
6.
Diffraction
Light Microscopy
A.
Basic features
B.
The compound light microscope
1.
Basic parts
a.
Eyepieces (ocular lens)
b.
Base
c.
Condenser
d.
Iris diaphragm
e.
Objective lens
f.
Body tube
g.
Mechanical stage
h.
Adjustment knobs
2.
Magnification
a.
Calculation
b.
Parfocal
3.
Microscopic measurement
C.
Dark-field microscopy
D.
Phase-contrast microscopy
E.
Nomarski (differential interference contrast) microscopy
E.
Fluorescence microscopy
1.
Fluorescent dyes
2.
Fluorescent antibody staining
F.
Confocal microscopy
G.
Digital microscopy
3-3
IV.
Electron Microscopy
A.
Basic features
B.
Transmission electron microscope (TEM)
1.
General features
2.
Shadow casting
a.
Freeze-fracturing
b.
Freeze-etching
3.
Electron micrographs
C.
Scanning electron microscope
1.
General features
2.
Scanning electron micrographs
D.
Scanning Tunneling Electron Microscope
1.
General features
2.
Atomic force microscope
V.
Techniques of Light Microscopy
A.
Preparation of specimens for the light microscope
1.
Wet mounts
2.
Hanging drop
3.
Smears
4.
Heat fixation
B.
Principles of staining
1.
Stains
a.
Cationic (basic) dyes
b.
Anionic (acidic) dyes
2.
Types of stains
a.
Simple stains
b.
Differential stains
3.
The Gram stain
a.
Basic steps
b.
Gram-variable reactions
4.
The Ziehl-Neelsen Acid-Fast Stain
5.
Special staining procedures
a.
Negative staining
b.
Flagellar staining
c.
Endospore staining
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