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BIOL 223 Lab#4
Structural Stains (Endospore and Capsule) and Staining Review
Objectives
After completing this exercise you should be able to prepare and interpret endospore and capsule
stains.
Background
Structural stains can be used to identify and study the structure of bacteria. Currently, most of
the fine structural details are examined using an electron microscope, but, historically, staining
techniques have given much insight into bacterial fine structure. We will examine a few
structural stains that are still useful today. These stains are used to observe endospores, capsules
and flagella. We will focus on endospore and capsule stains in this lab. See page 70 in
Microbiology, 8th Edition, Tortora, Funke and Case for illustrations of capsule and endospore
stains of bacteria.
Endospores
Endospores are formed by members of ten genera included in Bergey's Manual, endosporeforming gram-positive rods and cocci. Bacillus and Clostridium are the most familiar
genera. Endospores are called "resting bodies" because they do not metabolize and are resistant
to heating, various chemicals and many harsh environmental conditions. Endospores are not for
reproduction; they are formed when essential nutrients or water is not available. Once an
endospore forms inside a cell, the cell disintegrates. Endospores can remain dormant for long
periods of time. However, an endospore may return to its vegetative or growing state.
Taxonomically, it is very helpful to know whether a bacterium is an endospore former and also
the position of the endospores. Endospores are impermeable to most stains, so heat is used to
drive the stain into the endospore. Once stained, the endospores do not readily re-colorize. We
will use the Schaffer-Fulton endospore stain.
Capsules
Many bacteria secrete chemicals that adhere to their surfaces, forming a viscous coat. This
structure is called a capsule when it is round or oval in shape, and a slime layer when it is
irregularly shaped and loosely bound to the bacterium. The ability to form capsules is
genetically determined, but the size is influenced by the medium on which the bacterium is
growing. Most capsules are composed of polysaccharides, which are water-soluble and
uncharged. Because of the capsules nonionic nature, simple stains will not adhere to it. Most
capsule staining techniques stain the bacteria and the background, leaving the capsules
unstained-essentially a "negative" capsule stain.
Capsules have an important role in the virulence (disease-causing ability) of some bacteria. For
example, when bacteria such as Streptococcus pneumoniae have a capsule, the body's white
blood cells cannot phagocytize the bacteria efficiently, and disease occurs. When S. pneumoniae
lack a capsule, they are easily engulfed and are not virulent.
Materials
Slides
Paper towels
Wash bottle of distilled water
Endospore stain reagents: malachite green and safranin
Capsule stain reagents: Congo-red, acid-alcohol, and acid fuchsin
Cultures
Endospore stain
Bacillus megaterium (24-hour)
Bacillus subtilis (24-hour)
Bacillus subtilis (72-hour)
Capsule stain
Streptococcus salivarius
Escherichia coli
Biol 223 Spring 2005 Page 1 of 2
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