Exercise 6-D
STAINING OF MICROORGANISMS
ENDOSPORE STAINS, CAPSULE STAINS
& FLAGELLA
Introduction
Endospore stains, capsule stains, and flagellar stains are staining techniques that allow for the
differentiation of specific bacterial structures found either inside or outside of cells. Such stains are
sometimes referred to as special stains or structural stains. Bacterial endospores are dormant
structures produced by a variety of bacterial species and which are highly resistant to heat, desiccation,
and toxic chemicals. Endospores resist staining, but once stained are highly resistant to being
decolorized and counterstained (much like acid-fast cells). A single bacterium can contain only one
endospore, the shape and location of which is usually characteristic of the species. The degree to which
an endospore changes the shape of the sporangium (spore-containing cell) is also a feature useful in
identification. Not all cells present within a population will contain endospores, and older cells will
often degenerate leaving their spores (now called exospores) behind in the environment.
Many prokaryotic cells can produce a layer of gelatinous or slimy material external to their cell wall
referred to as a glycocalyx. If this material is highly organized into a compact structure it is called a
capsule and can be rendered visible with a capsule stain. If it is less well organized and tends to spread
into the surrounding medium it is called a slime layer and is less readily stained and observed. Capsules
may be made up of a variety of materials (polysaccharides, glycoproteins or polypeptides) but in general
appear to be water soluble, and are unstable when subjected to heat. The ability of bacteria to produce
capsules is genetically determined; however, individual cells may or may not have a visible glycocalyx
depending upon their age and the availability of necessary nutrients. Capsule stains are useful in
determining the virulence of pathogens, but are not overly useful for culture identification.
Bacterial flagella are normally too small to be observed with the light microscope; however, certain
staining techniques can render them visible. The procedures used in flagella staining involve covering
the surface of the flagella with a mordant, thus increasing their diameter. Although we will not make
flagellar stain preparations, we will observe some prepared slides of flagellar stains.
Methods:
A. Endospore Stain
The presence of endospores may be demonstrated using either of the two procedures (methods)
outlined below. In general the production of endospores by a bacterial culture requires time, so
the best spore samples will be obtained from cultures that are several days old. Many genera of
bacteria including Bacillus, Lysinibacillus, Paenibacillus, Brevibacillus and Clostridium are
known to produce endospores; however, even cultures that are genetically capable of forming
endospores may not contain these structures if growing conditions do not stimulate spore
formation.
Note – Endospores are often visible in Gram stain preparations. Under these circumstances,
mature endospores will typically appear as white or light colored areas within darker colored
cells; immature or germinating endospores may appear dark.
