Spore Stain (Schaeffer-Fulton Method)

& Capsule Stain

M-265 General Microbiology lab

Yasmine Al-Shboul

Although endospores were observed and reported by numerous scientists (including Perty, 1852; Pasteur, 1869; Koch, 1876; and Cohn, 1872), these structures were primarily described as refractile bodies because they could not be stained using the typical compounds (carmine, methylene blue, carbol fuchsin, safranin, etc.) applied in early staining protocols. 

Once the endospores were understood to be a dormant form of certain bacteria that demonstrated a unique resistance to treatment with heat, research efforts emphasized control of the endospores to improve sterilization, prevent infection, and limit contamination of foods. 

• Spore Forming Bacteria:• Genus Clostridium. (Gram positive anaerobic bacteria)• Genus Bacillus. (Gram positive aerobic bacteria)

• The Spore is Composed of:• Spore Coat.• Spore Wall.• Spore Membrane.• The Matrix (contains: DNA, RNA, Ribosoms,…)

• The Spore is resistant to:• Excessive Heat, Freezing, Radiation, Desiccation, Chemical Agents

including common stains.

• The cross section of a bacterial spore,

• Shape: Spherical, Oval.• Location: Terminal, Sub-Terminal, Central.• Size: Swollen, Normal

• Examples:• Cl. botulinum: swollen oval sub-terminal endospore.• Cl. perfringes: large oval central or sub-terminal endospore.• Cl. tetani: spherical swollen terminal (drumstick) endospore.

• Bacillus spp.: oval non-swollen central endospore.

Variations in endospore morphology

)2 ,3 ,5 (terminal (1, 4) central endospore

• Clostridium perfringens• CLOSTRIDIUM PERFRINGENS IS A SPECIES OF CLOSTRIDIUM WHOSE

SPORES ARE WIDELY DISTRIBUTED IN SOIL. IT IS ONE OF THE MOST COMMON CAUSES OF FOOD POISONING, WHICH DEVELOPS AFTER EATING FOOD CONTAMINATED WITH THE SPORES OF THE BACTERIA. THESE SPORES CAN WITHSTAND COOKING, AND ONCE INSIDE THE INTESTINE THEY PRODUCE A TOXIN THAT RESULTS IN SEVERE ABDOMINAL CRAMPS, AND WATERY DIARRHEA. THE U.S. FOOD AND DRUG ADMINISTRATION STATES THAT IT TAKES 8 TO 22 HOURS FOR THE SYMPTOMS TO ARISE, BUT THE ILLNESS USUALLY RESOLVES AFTER ABOUT 24 HOURS. CLOSTRIDIUM PERFRINGENS IS ALSO THE BACTERIA THAT MOST OFTEN CAUSES GAS GANGRENE. THE INFECTION CAN OCCUR IN TRAUMATIC, CRUSHING WOUNDS, OR IN DEEP PUNCTURE WOUNDS WHERE THERE IS AN ABSENCE OF OXYGEN. THE DISEASE CAN BE FATAL IF NOT TREATED PROMPTLY.

• Clostridium botulinum• CLOSTRIDIUM BOTULINUM IS AN ORGANISM CAPABLE OF

PRODUCING A POWERFUL TOXIN THAT HAS EFFECTS THE NERVOUS SYSTEM. AMOUNTS AS SMALL AS A FEW NANOGRAMS OF TOXIN CAN CAUSE ILLNESS, ACCORDING TO THE U.S. FOOD AND DRUG ADMINISTRATION. EATING FOOD CONTAMINATED WITH THE TOXIN RESULTS IN FOOD-BORNE BOTULISM. FOODS MOST OFTEN ASSOCIATED WITH THIS CONTAMINATION ARE UNCOOKED, UNDERCOOKED, AND HOME- CANNED FOODS.

•

Clostridium tetani• CLOSTRIDIUM TETANI IS AN ORGANISM WHOSE SPORES CAN BE

FOUND IN SOIL ALMOST ANYWHERE IN THE WORLD. IT IS THE CAUSE OF THE DISEASE TETANUS, OR LOCKJAW, WHICH IS RARE IN THE UNITED STATES, AND OTHER DEVELOPED COUNTRIES. THIS BACTERIUM PRODUCES A TOXIN CALLED TETANOSPASMIN, ACCORDING TO THE NATIONAL CENTER FOR BIOTECHNOLOGY INFORMATION. THE TOXIN AFFECTS THE NERVOUS SYSTEM, PRODUCING SPASMS AND PARALYSIS OF THE MUSCLES. TETANUS HAS A HIGH DEATH RATE IF NOT PROPERLY TREATED.

• Spore Stain:• Primary stain: Malachite Green (Green).• Decolorizing Agent: Tap Water.• Counter-Stain: Safranin (Red).

• Procedure:• 1.  Prepare smears of organisms to be tested for endospores. • 2.  Heat fix the smears. • 3.  Cover the smears with a piece of absorbent paper cut to fit the slide and

place the slide on a wire gauze on a ring stand.   4.  Saturate the paper with malachite green and holding the Bunsen burner in the hand heat the slide until steam can be seen rising from the surface.  Remove the heat and reheat the slide as needed to keep the slide steaming for about five minutes.  As the paper begins to dry add a drop or two of malachite green to keep it moist, but don't add so much at one time that the temperature is appreciably reduced.  DO NOT OVERHEAT.  The process is steaming and not baking.

• 5.  Remove the paper with tweezers and rinse the slide thoroughly with tap water.

• 6.  Drain the slide and counterstain 1 min. with safranin. • 7.  Wash, blot, and examine. • 8.  The vegetative cells will appear red and the spores will appear green. •

• Spore stain results

Capsule Stain

• Capsule: gelatinous layer secreted by the cell that surrounds and adheres to the cell wall.

• Composition:• Polysaccharides, Glycoproteins, or Polypeptides.• Procedure:• Place a drop of Eosin on the end of a clean slide.• Mix bacterial inoculum with the stain• Spread the Bacteria-Eosin Mixture with a second slide.• Air Dry.

• 1.Use a loop to mix a drop of water, a drop of eosin and a small amount of Klebsiella pneumoniae together at the end of a slide

• 2. Use another slide to spread the smear like a blood smear.  (As the instructor will demonstrate.)  Allow the smear to air dry. DON'T HEAT FIX!

• .3Air Dry

• Result of capsule staining

• GOOD LUCK