6/21/13 Endospores - DEFINITION

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DEFINITION

Certain bacteria can form endospores in dry environments in a process calledsporulation. They are called endospores because the spores form within the cell.Endospores offer great advantages to bacterial cells because they are extremelyresistant to a number of harsh environments, including: heat, desiccation, radiation,chemicals, acids, and drying. The discovery of bacterial endospores was important tomicrobiology because it provided knowledge on proper methods for sterilization ofculture media, foods, and other perishable items. Many organisms form spores, butthe bacterial endospore is unique in its heat resistance capabilities. Yet, how areendospores so resistant to harsh environments? The answer lies in the structure of theendospore 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 withcalcium ions embedded in the spore coat. This cross linkage greatly contributes to theextreme resistance capabilities of the endospores because it creates a highlyimpenetrable barrier. The calcium DPA cross linkages compose 10% of the dryweight of the endospores.

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"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 theendospores success in dry environments. However, the low water concentration andgel cytoplasm contributes to the inactivity of cytoplasmic enzymes. The corecytoplasm is also one unit lower in pH than the vegetative cell, thus conferring acidicenvironment survival. SASPs, small acid soluble spore proteins, are formed duringsporulation 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 duringgermination, the process of converting a spore back to a vegetative cell.

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6/21/13 Endospores - RESISTANCE

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RESISTANCE

Just how resistant are endospores? Several documented cases have reported theastonishing ability for endospores to remain viable, or capable of germinating backinto vegetative cells. Below are a few of the more famous published evidence:

A suspension of Clostridium Aceticum was prepared in 1947 and thirty-four yearslater 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 UnitedKingdom. The debris was over 2,000 years old but a significant number of viableThermoactinomyes species were cultured. Thermoactinomyes spores were alsorecovered in a Minnesota lake over 7,000 years old. In 1995, a group of scientists reported the recovery of bacterial spores from the gut ofan extinct bee 25 - 40 million years old. The bee was allegedly trapped and preservedin amber. Samples of bee tissue incubated in sterile culture yielded endosporeforming bacteria. Scientist also claimed the isolation of halophilic bacterial endospores from saltcrystals of the Permian age, over 250 million years old.

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"250 Million Year Old Crystals Containing Viable Endospores" (6)

All of the above cases stressed the highest degree of no contamination from outsidesources.

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6/21/13 Endospores - THE STAIN

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THE STAIN

Procedure:

1) Smear the organism and heat fix to a slide2) Place the slide over a steam bath and cover with Malachite Green3) Keep the stain over the bath for 3 - 5 minutes, recovering the slide with MalachiteGreen if some evaporates4) Dump the Malachite Green off and allow to cool5) Rinse the slide with water to remove excess stain6) Cover the smear with Safranin for two minutes7) Rinse the slide with water to remove excess stain8) 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 highlyresistant 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

6/21/13 Endospores - THE STAIN

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degree of resistance due to multiple coats surrounding the spore. Therefore, duringthe primary stain, the smeared culture is heated over a steam bath to soften the sturdyand durable outer layer of the cell and allow the Malachite Green to bind within thespore.The most common staining procedure used is the Schaeffer-Fulton. This procedureuses Malachite Green as the primary stain for the spores. This stain can not bedecolorized from the spores once they are removed from the heat; however, water canremove the Malachite Green from the vegetative cells, making them again colorless.The vegetative cell is counterstained with Safranin. This causes the surroundingsporangium to appear red/pink.When the smear is removed from the heat and allowed to dry, the sporesharden/reseal their outer layer and trap the primary stain, Malachite Green, inside. Atthis point, the vegetative cells are also green. When the smear is washed, the primarystain is removed from the vegetative cells. Safranin, the counter stain, is then addedand only taken up by the vegetative cells. This is because the counter stain is appliedat 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.

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6/21/13 Endospores - DISEASES

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DISEASES

Clostridium tetaniCauses tetanus also known as lockjaw in humans. The spores may be obtained froma contaminated puncture. Spores germinate in anaerobic environments, and thebacteria release tetanospasmin in tissues. The exotoxin causes nervous systemabnormalities, constant skeletal muscle contraction, and may lead to respiratoryfailure.

Way to prevent disease: immunization

Laboratory Results: Motile, Terminal spores, Non-aerotolerant

Clostridium botulinumCauses botulism food poisoning. Air born spores infect food before canning or jarring,and the spores germinate in the canned anaerobic environment. Honey is the mostcommon 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 perfringensContracted from dirt through large cuts and wounds, the bacterium releases anecrosis-causing toxin. Producing a gas that deforms the infected tissue, theinfectious agent causes host to experience severe diarrhea.

Treatment: Penicillin G and antitoxin

Laboratory Results: Motile, No terminal spores, Non-aerotolerant, Possess double-zone hemolysis

Clostridium difficileThe bacterium may be found as natural intestinal flora. Infection occurs through usageof broad-spectrum antibiotics that kill other natural intestinal flora, allowing C. difficileto proliferate and infect the large intestine. The bacterium releases two enterotoxinsthat 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, contaminatedanimal products, or air-born spores. Inside the body, anthrax spores grow at anexponential rate and release an immunosuppressive toxin. Anthrax targets three

6/21/13 Endospores - DISEASES

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areas: skin, intestines, and lungs. Symptoms of skin infections commence with anitchy bump the size of a small bug bite that grows to form a liquid-filled sac in a one ortwo day period. The sac transforms in to an ulcer with a center consisting of blacknecrotic tissue. Symptoms of intestinal infections include nausea, lack of appetite, andvomiting. As the infection persists, symptoms worsen to consist of severe abdominalpain, vomiting of blood, and severe diarrhea. Symptoms of inhaled anthrax resembleflu-like symptoms that become more severe and consist of respiratory difficulty andshock.

"Bacillus anthracis" (8)

Treatment: dioxycyclin and ciprofloxacin**Anthrax vaccine is not recommended or available to the public. New treatments andvaccines 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

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