CHAPTER
4
Microscopy, Staining, and
Classification
Chapter Outline
Units of Measurement (p. 97)
Microscopy (pp. 97–106)
General Principles of Microscopy
Light Microscopy
Staining (pp. 106–112)
Preparing Specimens for Staining
Principles of Staining
Simple Stains
Differential Stains
Special Stains
Staining for Electron Microscopy
Classification and Identification of Microorganisms (pp. 112–119)
Linnaeus and Taxonomic Categories
Domains
Chapter Summary
Units of Measurement (p. 97)
Scientists use metric units of measurement that are simpler than English units and are
standardized throughout the world. The metric system is a decimal system in which each unit is
one-tenth the size of the next largest unit. The basic unit of length in the metric system is the
meter (m), which is slightly longer than an English yard. One-tenth of a meter is a decimeter,
Chapter 4 Microscopy, Staining, and Classification
Microscopy (pp. 97–106)
Microscopy refers to the use of light or electrons to magnify objects.
General Principles of Microscopy
The same general principles apply to both light and electron microscopy.
Wavelength of Radiation
Various forms of radiation differ in wavelength, which is the distance between two
corresponding parts of a wave (ray). The human eye distinguishes different wavelengths of light
Magnification
Magnification is the apparent increase in size of an object and is indicated by a number
followed by an “”, which is read “times.” Magnification results when a beam of radiation
refracts (bends) as it passes through a lens. Refraction occurs because the lens is optically dense
Resolution
Resolution (also called resolving power) is the ability to distinguish between objects that are
close together. The better the resolution, the better the ability to distinguish two objects that are
close to one another. Modern microscopes can distinguish between objects as close together as
0.2 µm. A principle of microscopy is that resolution distance is dependent on (1) the wavelength
of the electromagnetic radiation and (2) the numerical aperture of the lens, which is its ability
to gather light.
Contrast
Contrast refers to differences in intensity between two objects, or between an object and its
background. Since most microorganisms are colorless, they are stained to increase contrast. The
use of light that is in phase may also be used to enhance contrast.
Light Microscopy
Several classes of microscopes use various types of light to examine specimens.
Bright-Field Microscopes
The most common microscopes are bright-field microscopes, in which the background (or field)
is illuminated. There are two basic types: Simple microscopes contain a single magnifying lens
Instructor’s Manual for Microbiology with Diseases by Body Systems, 5e
objective lenses are mounted on a revolving nosepiece. Objective lenses on a typical
microscope include a scanning objective (4×), a low power objective (10×), a high dry objective
(40×) and an oil immersion objective (100×) lens. An oil immersion lens increases the
magnification and the resolution. Immersion oil is used to fill the space between the specimen
Dark-Field Microscopes
Pale objects are best observed with dark-field microscopes. Light rays are reflected inside the
condenser so the light passes into the slide at an oblique angle. Only light rays scattered by the
specimen enter the objective lens and are seen, so the specimen appears light against a dark
background. These microscopes are useful for observing small and colorless cells.
Phase Microscopes
Phase microscopes are used to observe living cells or unfixed, unstained specimens. Contrast is
produced by treating sets of light rays differently, then bringing them together again. Phase-
Fluorescence Microscopes
Molecules that absorb invisible ultraviolet light then emit longer visible wavelengths are
fluorescent. Fluorescence microscopes use an ultraviolet (UV) light source to cause objects to
fluoresce. Because UV light has a shorter wavelength than visible light, resolution is increased.
Confocal Microscopes
Confocal microscopes use fluorescent dyes in conjunction with ultraviolet lasers to illuminate
the fluorescent chemicals in only one thin plane of a specimen at a time. Several images are
Chapter 4 Microscopy, Staining, and Classification
Electron Microscopy
Because the shortest wavelength of visible light is about 400 nm, structures closer together than
about 200 nm cannot be distinguished using light microscopy. By contrast, electrons traveling as
Transmission Electron Microscopes
A transmission electron microscope (TEM) generates a beam of electrons that passes through
a thinly sliced, dehydrated specimen, through magnetic fields that manipulate and focus the
beam, and then onto a fluorescent screen that changes the electrons’ energy into visible light.
High density areas in the specimen block electrons, producing dark areas, while low density
Scanning Electron Microscopes
In a scanning electron microscope (SEM), the surface of the specimen is first coated with a
metal such as platinum or gold. The SEM then focuses the beam of electrons back and forth
across the surface of the coated specimen, scanning it rather than penetrating it. Electrons
Probe Microscopy
Probe microscopes use miniscule electronic probes to magnify specimens more than
100,000,000. There are two types. Scanning tunneling microscopes (STMs) pass a pointed
metallic probe across and above the surface of a specimen and measure the amount of electron
Staining (pp. 106–112)
Both light and electron microscopy use staining—the coloring of specimens with dyes—to
increase contrast and resolution.
Preparing Specimens for Staining
Preparing specimens for staining involves making a thin film of organisms—or smear—of the
specimen on a slide, and fixing it to the slide. The smear can be fixed by either passing the slide
Instructor’s Manual for Microbiology with Diseases by Body Systems, 5e
Principles of Staining
Microbiological dyes are usually salts in which either the positively charged cation or negatively
charged anion is colored. The colored portion of a dye, known as the chromophore, typically
binds to chemicals via covalent, ionic, or hydrogen bonds. Anionic chromophores called acidic
Simple Stains
Simple stains are composed of a single basic dye such as crystal violet and involve no more
than soaking the smear in the dye and rinsing.
Differential Stains
Differential stains use more than one dye so that different cells, chemicals, or structures can be
distinguished.
The Gram stain differentiates between purple-staining Gram-positive cells and pink-staining
Gram-negative cells, which differ in the structure of their cell walls. The classical procedure has
four steps:
2. Flood the smear with the mordant, iodine for one minute, and rinse.
4. Flood the smear with the counterstain, safranin, for one minute and rinse.
(1) Cover the smear with tissue paper. (2) Flood the slide with carbolfuchsin for several
minutes while steam-heating the slide. (3) Rinse the cooled slide with hydrochloric acid and
alcohol. (4) Counterstain with methylene blue. The presence of pink acid-fast bacilli (AFBs) in
sputum is an indication of mycobacterial infection.
Endospores cannot be stained by normal techniques because their walls are practically
Special Stains
Special stains reveal specific structures. Fluorescent stains discussed earlier are examples.
Acidic dyes are repulsed by the negative charges on the surface of cells and therefore do not
stain them. Such dyes that stain the background and leave the cells colorless are called negative
Copyright © 2018 Pearson Education, Inc. 29
Staining for Electron Microscopy
Stains used for TEM are chemicals containing atoms of heavy metals, such as lead, which
Classification and Identification of Microorganisms
(pp. 112–119)
Biologists sort organisms on the basis of mutual similarities into nonoverlapping groups called
taxa. Taxonomy is the science of classification, nomenclature (rules of naming), and
Linnaeus and Taxonomic Categories
Carolus Linnaeus invented a standardized system of taxonomy, grouping similar interbreeding
organisms into species. Since most microbes do not interbreed, some scientists use the term
Domains
Carl Woese and George Fox proposed the existence of three taxonomic domains based on three
cell types revealed by rRNA sequencing: Eukarya, Bacteria, and Archaea. Cells of the three
Taxonomic and Identifying Characteristics
Taxonomists use both macroscopic and microscopic examination of microbes as well as other
1. Many physical characteristics are used to identify microorganisms. For example, scientists
2. Microbiologists also use biochemical tests, noting a particular microbe’s ability to utilize or
3. Many microbes are antigenic, triggering the production of antibodies. Serological tests using
antiserum (serum containing antibodies) can determine whether a microorganism produces
4. Bacteriophages (or simply phages) are viruses that infect and usually destroy bacterial cells.
Bacteria are spread across a plate and drops containing phage solutions are placed at specific
5. Scientists also analyze a specimen’s nucleic acid content and sequence. DNA sequencing and
Taxonomic Keys
Microbiologists use dichotomous keys, which involve stepwise either/or choices between
paired characteristics, to assist in identifying microorganisms.
New Media Resources
MicroMatters: Make the connection between Chapters 2, 3, and 4.
Learning Catalytics: Assess students in real time using open-ended tasks to probe student
understanding.