6/21/13
HOME
DEFINTION
STAIN
RESISTANCE
DISEASES
REFERENCES
Endospores - DEFINITION
DEFINITION
Certain bacteria can form endospores in dry environments in a process called
sporulation. They are called endospores because the spores form within the cell.
Endospores offer great advantages to bacterial cells because they are extremely
resistant to a number of harsh environments, including: heat, desiccation, radiation,
chemicals, acids, and drying. The discovery of bacterial endospores was important to
microbiology because it provided knowledge on proper methods for sterilization of
culture media, foods, and other perishable items. Many organisms form spores, but
the bacterial endospore is unique in its heat resistance capabilities. Yet, how are
endospores so resistant to harsh environments? The answer lies in the structure of the
endospore illustrated below:
"Endospore Structure" (3)
Spore Structure:
Exosporium - A thin delicate covering made of protein.
Spore coats - Composed of layers of spore specific proteins.
Cortex - Composed of loosely linked peptidoglycan and contains dipicolinic acid
(DPA), which is particular to all bacterial endospores. The DPA cross links with
calcium ions embedded in the spore coat. This cross linkage greatly contributes to the
extreme resistance capabilities of the endospores because it creates a highly
impenetrable barrier. The calcium DPA cross linkages compose 10% of the dry
weight of the endospores.
pscantie.myweb.uga.edu/definition.html
1/2
6/21/13
Endospores - DEFINITION
"Calcium Endospore Cross Linkages Confer Resistance" (4)
Core - The core contains the usual cell wall and, cytoplasmic membrane, nucleoid,
and cytoplasm. The core only has 10-30% of the water content of vegetative cells;
therefore the core cytoplasm is in a gel state. The low water content contributes to the
endospores success in dry environments. However, the low water concentration and
gel cytoplasm contributes to the inactivity of cytoplasmic enzymes. The core
cytoplasm is also one unit lower in pH than the vegetative cell, thus conferring acidic
environment survival. SASPs, small acid soluble spore proteins, are formed during
sporulation and bind to DNA in the core. SASPs protect the DNA from UV light,
desiccation, and dry heat. SASPs also serve as a carbon energy source during
germination, the process of converting a spore back to a vegetative cell.
HOME
pscantie.myweb.uga.edu/definition.html
| DEFINTION | STAIN | RESISTANCE | DISEASES | REFERENCES
2/2
6/21/13
HOME
DEFINTION
STAIN
RESISTANCE
DISEASES
REFERENCES
Endospores - RESISTANCE
RESISTANCE
Just how resistant are endospores? Several documented cases have reported the
astonishing ability for endospores to remain viable, or capable of germinating back
into vegetative cells. Below are a few of the more famous published evidence:
A suspension of Clostridium Aceticum was prepared in 1947 and thirty-four years
later cultured onto nutrient agar media; growth commenced in less than 12 hours.
"34 Year Old Clostridium Spores" (5)
Microbiologists examined debris from a Roman archeological site in the United
Kingdom. The debris was over 2,000 years old but a significant number of viable
Thermoactinomyes species were cultured. Thermoactinomyes spores were also
recovered in a Minnesota lake over 7,000 years old.
In 1995, a group of scientists reported the recovery of bacterial spores from the gut of
an extinct bee 25 - 40 million years old. The bee was allegedly trapped and preserved
in amber. Samples of bee tissue incubated in sterile culture yielded endospore
forming bacteria.
Scientist also claimed the isolation of halophilic bacterial endospores from salt
crystals of the Permian age, over 250 million years old.
pscantie.myweb.uga.edu/resistance.html
1/2
6/21/13
Endospores - RESISTANCE
"250 Million Year Old Crystals Containing Viable Endospores" (6)
All of the above cases stressed the highest degree of no contamination from outside
sources.
HOME
pscantie.myweb.uga.edu/resistance.html
| DEFINTION | STAIN | RESISTANCE | DISEASES | REFERENCES
2/2
6/21/13
HOME
DEFINTION
STAIN
RESISTANCE
DISEASES
REFERENCES
Endospores - THE STAIN
THE STAIN
Procedure:
1) Smear the organism and heat fix to a slide
2) Place the slide over a steam bath and cover with Malachite Green
3) Keep the stain over the bath for 3 - 5 minutes, recovering the slide with Malachite
Green if some evaporates
4) Dump the Malachite Green off and allow to cool
5) Rinse the slide with water to remove excess stain
6) Cover the smear with Safranin for two minutes
7) Rinse the slide with water to remove excess stain
8) Blot dry the stain and view under a microscope
"Smear Diagram" (7)
Step Finished
Color of Vegetative Cell Color of Endospore
Smear
Colorless
Colorless
Malachite Green Green
Green
Cool/Wash
Colorless
Green
Safranin
Safranin
Green
Why This Stain:
The Endospore stain is a special stain that is used to see specialized cell structures.
The Endospore stain is used to determine if certain bacterium cells contain highly
resistant spores within their vegetative cells.
How it Works:
It is difficult to get a dye into an endospore because of its low penetrability and high
pscantie.myweb.uga.edu/stain.html
1/2
6/21/13
Endospores - THE STAIN
degree of resistance due to multiple coats surrounding the spore. Therefore, during
the primary stain, the smeared culture is heated over a steam bath to soften the sturdy
and durable outer layer of the cell and allow the Malachite Green to bind within the
spore.
The most common staining procedure used is the Schaeffer-Fulton. This procedure
uses Malachite Green as the primary stain for the spores. This stain can not be
decolorized from the spores once they are removed from the heat; however, water can
remove the Malachite Green from the vegetative cells, making them again colorless.
The vegetative cell is counterstained with Safranin. This causes the surrounding
sporangium to appear red/pink.
When the smear is removed from the heat and allowed to dry, the spores
harden/reseal their outer layer and trap the primary stain, Malachite Green, inside. At
this point, the vegetative cells are also green. When the smear is washed, the primary
stain is removed from the vegetative cells. Safranin, the counter stain, is then added
and only taken up by the vegetative cells. This is because the counter stain is applied
at room temperature and the spores are tightly closed and full of Malachite Green.
Once completely dried, the vegetative cells are red/pink and the spores are green.
HOME
pscantie.myweb.uga.edu/stain.html
| DEFINTION | STAIN | RESISTANCE | DISEASES | REFERENCES
2/2
6/21/13
HOME
DEFINTION
STAIN
RESISTANCE
DISEASES
REFERENCES
Endospores - DISEASES
DISEASES
Clostridium tetani
Causes tetanus also known as lockjaw in humans. The spores may be obtained from
a contaminated puncture. Spores germinate in anaerobic environments, and the
bacteria release tetanospasmin in tissues. The exotoxin causes nervous system
abnormalities, constant skeletal muscle contraction, and may lead to respiratory
failure.
Way to prevent disease: immunization
Laboratory Results: Motile, Terminal spores, Non-aerotolerant
Clostridium botulinum
Causes botulism food poisoning. Air born spores infect food before canning or jarring,
and the spores germinate in the canned anaerobic environment. Honey is the most
common infective food source for children; however, juvenile botulism is not as deadly.
Symptoms include muscular paralysis and blurred vision.
Treatment: anti-toxin
Laboratory Results: Motile, No terminal spores, Non-aerotolerant, Lipase (+)
Clostridium perfringens
Contracted from dirt through large cuts and wounds, the bacterium releases a
necrosis-causing toxin. Producing a gas that deforms the infected tissue, the
infectious agent causes host to experience severe diarrhea.
Treatment: Penicillin G and antitoxin
Laboratory Results: Motile, No terminal spores, Non-aerotolerant, Possess doublezone hemolysis
Clostridium difficile
The bacterium may be found as natural intestinal flora. Infection occurs through usage
of broad-spectrum antibiotics that kill other natural intestinal flora, allowing C. difficile
to proliferate and infect the large intestine. The bacterium releases two enterotoxins
that harm the intestinal lining and cause diarrhea.
Treatment: oral vancomycin
Laboratory Results: Motility, No terminal spores, Non-aerotolerant
Bacillus anthracis
Found in soil, infection may occur through contact with infected animals, contaminated
animal products, or air-born spores. Inside the body, anthrax spores grow at an
exponential rate and release an immunosuppressive toxin. Anthrax targets three
pscantie.myweb.uga.edu/diseases.html
1/2
6/21/13
Endospores - DISEASES
areas: skin, intestines, and lungs. Symptoms of skin infections commence with an
itchy bump the size of a small bug bite that grows to form a liquid-filled sac in a one or
two day period. The sac transforms in to an ulcer with a center consisting of black
necrotic tissue. Symptoms of intestinal infections include nausea, lack of appetite, and
vomiting. As the infection persists, symptoms worsen to consist of severe abdominal
pain, vomiting of blood, and severe diarrhea. Symptoms of inhaled anthrax resemble
flu-like symptoms that become more severe and consist of respiratory difficulty and
shock.
"Bacillus anthracis" (8)
Treatment: dioxycyclin and ciprofloxacin
**Anthrax vaccine is not recommended or available to the public. New treatments and
vaccines are under development**
Chances of survival:
Skin infections: kill 20% of infected people
Intestinal infections: kill 25-60% of infected people
Inhaled infections: often fatal
Information obtained from:
http://medic.med.uth.tmc.edu/path/00001496.htm
HOME
pscantie.myweb.uga.edu/diseases.html
| DEFINTION | STAIN | RESISTANCE | DISEASES | REFERENCES
2/2