Special stains

Special stains

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  SPECIAL STAINS Dr. DineshKr Jain, MD., Assistant professor, Department of Microbiology,SMS Medical college, Jaipur
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  SPECIAL STAINS I. STAININGOF FLAGELLA II. STAINING OF CAPSULE III. STAINING OF ENDOSPORE
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  FLAGELLAR STAINS • Tovisualize the presence and arrangement of flagella for the presumptive identification of motile bacterial species. • Most motile bacteria possess flagella, the shape, number, and position of which are important in the identification, particularly when biochemical reactions are weak or equivocal.
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  PRINCIPLE • The Leifsonstaining technique (or modification) is most commonly used in clinical laboratories and is not difficult to perform, providing exact details that are followed in each step of the procedure. • Bacterial flagella can be stained by alcoholic solutions of rosaniline dyes that form a precipitate as the alcohol evaporates in the staining procedure. Basic fuchsin (pararosaniline acetate) serves as the primary stain with tannic acid added to the solution as a mordant. A counterstain, such as methylene blue, may be used to better visualize the bacteria in instances in which the primary stain is weak or does not react at all with the bacterial cell wall.
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  METHOD 1. LEIFSON METHOD Mostcommonly used Method • Reagents 1. Primary Stain- Basic fuchsin (para rosaniline acetate) 1.2% in 95% alcohol 2. Mordant-tannic acid 3% in water 3. Sodium chloride, 1.5% in water 4. Final Stain-1:1:1 Counterstain-methylene blue for better visualization • Stain for 5–15 minutes by alcoholic solutions of rosaniline dyes , allowing a precipitate to form as the alcohol evaporates • Observe the stained slide under the oil-immersion (100×) objective of the microscope -staining red to blue- black flagella should be observed
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  2. RYU METHOD Easyto perform and gives good results • Solution I- a. 5% phenol, 10.0 ml b. Powdered tannic acid, 2.0 g c. Saturated aluminum potassium sulfate 12-hydrate (crystals) • Solution II- Saturated solution of crystal violet in alcohol • Final Stain: Solution I: II in 10:1 ratio Method • Flood the air-dried smears with the staining solution for 1–5 minutes. • Wash the staining solution off in tap water. After the smears have dried, examine them under the oil-immersion objective of the microscope. Cell bodies & flagella stain violet.
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  3. Wet-Mount Technique Heimbrookand colleagues have described use of the wet-mount technique of Mayfield and Innis and the stain of Ryu as a rapid, simple way of staining flagella. • Bacteria grown on a non inhibitory medium for 16 to 24 hours is touched by wire and then touching a drop of water on a slide. • A coverslip is placed over the drop of a faintly turbid suspension, and the slide is examined for motile cells. • After 5 to 10 minutes, or when about half of the cells are attached to the glass slide or coverslip, two drops of the Ryu stain are applied to the edge of the coverslip and allowed to flow under the coverslip by capillary action. • The cells are examined for the presence of flagella after 5-15 minutes at room temperature.
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  INTERPRETATION • Look formorphology of flagella, the shape, number, and position • Polar • Monotrichous—single flagellum at one or both poles • Multitrichous—two or more flagella at one or both poles • Subpolar—flagella near pole with base of flagella at right angle to long axis • Lateral—flagella projecting from the middle of bacterial cell • Peritrichous—flagella haphazardly arranged all around bacterial cell • Positive control peritrichous, Escherichia coli; polar, monotrichous, Pseudomonas aeruginosa; multitrichous, Burkholderia cepacia • Negative Control Acinetobacter baumannii
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  Alcaligenes spp., peritrichous flagella Pseudomonasaeruginosa, polar flagella
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  STAINING OF CAPSULE PRINCIPLE Bacterialcapsules are non ionic so neither acidic or basic stains will adhere to their surfaces. Therefore the best way to visualize them is to stain the background using acidic stain and to stain the cell itself using a basic stain. Example : India ink which leaves the capsule as a clear halo surrounding a purple cell in a black field.
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  Negative staining byIndia ink and Nigrosin stain • Bacteria are mixed on a slide with an acidic dye such as congo red or the black stain, India ink, Nigrosin. The mixture is smeared across the face of the slide and allowed to air dry. • Because the stain carries a negative charge, it is repelled by the bacteria, which also have a negative charge. The stain gathers around the cell. Since a chemical reaction has not taken place, and because heat fixing has been avoided, the cells appear less shriveled or distorted. They often appear larger than stained cells and more natural. • India ink – aqueous solution of fine carbon particles (unable to penetrate the cells).
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  Negative staining byIndia ink and Nigrosin stain: • Capsule appears as a clear refractile halo around the bacteria where as both the bacteria and the background appear black. • India Ink preparation, illustrating the irregular- sized,encapsulated, spherical yeast cells of Cryptococcus neoformans
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  M'FAYDEAN CAPSULE STAIN •. • It is used for demonstration of capsule of Bacillus anthracis by using polychrome methylene blue stain. • M fadyean's reaction- amorphous purple capsule surrounding blue bacilli (Polychrome Methylene Blue Stain)
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  Quellung reaction This microscopic“precipitin test” can be used to identify pneumococci or to determine the capsular serotype of individual pneumococcal isolates by adding antisera mixed with Methylene Blue. • Reaction of the anti-capsular antibodies with the carbohydrate material of the capsule causes a micro precipitin reaction on the surface of the organism and a change in the refractive index of the capsule itself. • A small amount of methylene blue is added to the preparation to allow visualization of the cells and to provide contrast so that subtle refractile changes in the capsule can be more easily discerned. Microscopically, the capsule appears to “swell.”
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  STAINING OF ENDOSPORE •The spores are highly resistant to normal staining procedures due to their tough protein coats. • The most commonly used method is Schaeffer-Fulton method. • Principle: The primary stain in endospore staining procedures (malachite green) is driven into the cells with heat since malachite green is water soluble and doesn’t adhere well to the cell & since vegetative cells have been disrupted by heat, malachite green rinses easily from the vegetative cells allowing them to readily take up the counterstain.
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  Place dried &heat fixed slide over beaker of boiling water with bacterial film uppermost Within seconds, condensation of droplets on underside of slide; pour 5% aqueous solution of malachite green & leave for 1 mins with water continue to boil. Wash in cold water & treat it with 0.5% safranin or 0.05% basic fuchsin for 30 seconds.Wash & dry Observe it under microscope
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  Schaeffer-Fulton Stain
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  Method Primary StainDecoloriser Counterstain Interpretation Grams Stain Crystal Violet Acetone Safranin Spore-colorless Bacteria-Violet Modified Ziehl- Neelsen Staim Carbol Fuchsin 0.25-0.5% Sulphuric acid Loeffler’s Methylene Blue Spore-red Bacteria-Blue Dorner Stain Carbol Fuchsin Acid Alcohol Nigrosin Spore-red Bacteria-colorless Variation in Dorner Stain Carbol Fuchsin Nigrosin Spore-red Bacteria-colorless Schaeffer-Fulton Stain Malachite Green Water Safranin Spore-green Bacteria-red Bartholomew & Mittwer Method Malachite Green Water Safranin Spore-green Bacteria-red Abbot’s Method Methylene Blue Acid Alcohol Aniline-fuchsin Spore-Blue Bacteria-red Moeller Stain Carbol Fuchsin Acidified Ethanol Methylene Blue Spore-red Bacteria-Blue Modified Moeller Stain Kinyoun’s Carbol- fuchsin Kinyoun’s carbol-fuchsin Loeffler’s Methylene Blue Spore-red Bacteria-blue
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  REFRENCES • Bailey &Scott’s Diagnostic Microbiology • Koneman’s color atlas and textbook 7th Edition • Mackie & McCartney Practical Medical Microbiology 14th Edition
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  THANK YOU 13th September,1853 166th Birth-Anniversary of Christian Gram