The Genus Bacillus (page 3)
(This chapter has 6 pages)
© Kenneth Todar, PhD
Endospores were first described by Cohn in Bacillus
and later by Koch in the pathogen, Bacillus anthracis. Cohn
the heat resistance of endospores in B. subtilis, and Koch
the developmental cycle of spore formation in B. anthracis. Endospores
are so named because they are formed intacellularly, although they are
eventually released from this mother cell or sporangium as free spores.
Endospores have proven to be the most durable type of cell found in
and in their cryptobiotic state of dormancy they can remain viable for
extremely long periods of time, perhaps millions of years.
When viewed unstained, endospores of living bacilli appear edged in
black and are very bright and refractile. Endospores strongly resist
of simple stains or dyes and hence appear as nonstaining entities in
preparations. However, once stained, endospores are quite resistant to
decolorization. This is the basis of several spore stains such as the
staining method which also differentiates the spores from
and vegetative cells.
phase micrograph. Endospores can be readily recognized microscopically
by their intracellular site of formation and their extreme
Right. Bacillus anthracis Crystal violet stain viewed by light
Endospores are highly resistant to application of basic aniline dyes
readily stain vegetative cells.
Below. Spore stain of a Bacillus
species. CDC. The staining technique employed is the Schaeffer-Fulton
A fixed smear is flooded with a solution of malachite green and placed
over boiling water for 5 minutes. After rinsing, the smear is
with safranine. Mature spores stain green, whether free or still in the
vegetative sporangium; vegetative cells and sporangia stain red.
Endospores do not form normally during active growth and cell
Rather, their differentiation begins when a population of vegetative
passes out of the exponential phase of growth, usually as a result of
depletion. Typically one endospore is formed per vegetative cell. The
spore is liberated by lysis of the mother cell (sporangium) in which it
The formation of endospores
is a complex and highly-regulated form of development in a relatively
(procaryotic) cell. In all Bacillus species studied, the
of spore formation is similar, and can be divided into seven defined
(0-VI). The vegetative cell (a) begins spore development when the DNA
along the central axis of the cell as an "axial filament" (b).
DNA then separates and one chromosome becomes enclosed in plasma
to form a protoplast (c). The protoplast is then engulfed by the mother
cell membrane to form a intermediate structure called a forespore
(d) . Between the two membranes, The core (cell) wall, cortex and spore
coats are synthesized (e). As water is removed from the spore and as it
matures, it becomes increasingly heat resistant and more refractile
The mature spore is eventually liberated by lysis of the mother cell.
entire process takes place over a period of 6-7 hours and requires the
temporal regulation of more than 50 unique genes. Pasteur Institute.
Mature spores have no detectable metabolism, a state that is
described as cryptobiotic. They are highly resistant to
stresses such as high temperature (some endospores can be boiled for
hours and retain their viability), irradiation, strong acids,
etc. Although cryptobiotic, they retain viability indefinitely such
under appropriate environmental conditions, they germinate into
cells. Endospores are formed by vegetative cells in response to
signals that indicate a limiting factor for vegetative growth, such as
exhaustion of an essential nutrient. They germinate and become
cells when the environmental stress is relieved. Hence,
is a mechanism of survival rather than a mechanism of reproduction.
Below. Drawing of a
of a Bacillus endospore by Viake Haas, University of Wisconsin.
In cross section, Bacillus spores show a more complex
than that seen in vegetative cells. The spore protoplast (core) is
by the core (cell) wall, the cortex, and then the spore coat. Depending
on the species, an exosporium may be present. The core wall is composed
of the same type of peptidoglycan as the vegetative cell wall. The
is composed of a unique peptidoglycan that bears three repeat subunits,
always contains DAP, and has very little cross-linking between
chains. The outer spore coat represents 30-60 percent of the dry weight
of the spore. The spore coat proteins have an unusually high content of
cysteine and of hydrophobic amino acids, and are highly resistant to
that solubilize most proteins.
Table 3. Differences
and vegetative cells that form them.
wall polymer; crystalline S-layer
||Thick spore coat,
cortex, and unique
peptidoglycan core wall; no S-layer
|Calcium dipicolinic acid
||Present in core
|Resistance to chemicals
|Sensitivity to lysozyme
||Some sensitive; some
|Sensitivity to dyes and
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