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Similar to acid-faststaining for differentiate acid fast bacteria.pptx
acid-faststaining for differentiate acid fast bacteria.pptx
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- 2. Acid-fast staining • isa differential staining • used to differentiate acid fast and non acid fast bacteria. • used to identify acid-fast organisms such as members of the genus Mycobacterium . • also known as Ziehl-Neelsen method • first introduced by Paul Ehrlich later modified by two German doctors bacteriologist Franz Ziehl (1859–1926) and the pathologist Friedrich Neelsen (1854–1898). A.B
- 3. What are acidfast bacteria? • those which have a high content of mycolic acids in their cell walls. • are characterized by • wax-like, nearly impermeable cell walls; • contain mycolic acid and large amounts of fatty acids, waxes, and complex lipids. • are highly resistant to disinfectants and dry conditions. • Due to their waxy cell wall components, Mycobacterium are acid fast; that is, they retain the red dye, carbol fuchsin, after rinsing with acid solvents. A.B
- 4. Acid fast organisms Acid-fastorganisms like Mycobacterium contain large amounts of lipid substances,mycolic acids. which resist staining by ordinary methods such as a Gram stain. Require acid-fast staining that is used to screen for the Mycobacterium, Nocardia, and Legionella species in body tissues and fluids. Acid-fastness is an uncommon characteristic shared by the genera Mycobacterium and Nocardia (weakly acid-fast). Because of this feature, this stain is extremely helpful in identification in diseases caused by acid-fast bacteria, particularly tuberculosis and leprosy. A.B
- 5. Principle • Because thecell wall containing mycolic acid is so waxy and resistant to most compounds, acid-fast organisms require a special staining technique. • The primary stain used in acid-fast staining, carbol fuchsin, is lipid-soluble and contains phenol, which helps the stain penetrate the cell wall. This is further assisted by the addition of heat. • The smear is then rinsed with a very strong decolorizer, which strips the stain from all non-acid-fast cells but does not permeate the cell wall of acid-fast organisms. • The decolorized non-acid-fast cells then take up the counterstain. • Acid fast bacteria will be red, while nonacid fast bacteria will stain green. A.B
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- 7. Requirements Reagents • Primary Stain:Carbol-fuchsin. • Decoloriser: (HCl+Ethanol) • Counterstain: Methylene blue. Materials Slides Sample Inoculating loop Bunsen burner Cotton Microscope A.B
- 8. Preparation of reagents •Primary Stain: 0.3% Carbol-fuchsin. • Dissolve 50 g phenol in 100 mL ethanol (95%). • Dissolve 3 g Basic fuchsin in the mixture and add distilled water to bring the volume to 1 L. Decolorization Solution: (HCl+Ethanol) • Add 30 mL hydrochloric acid to 1 L of 95% denatured alcohol. • Cool and mix well before use. Counterstain: 0.3% Methylene blue. • Dissolve 3 g methylene blue in 1 L distilled water. A.B
- 9. Procedure • Take aclean grease free slide. • Prepare the smear from provided sample. • Air dry and heat fixed the smear. • Place a small strip of filter paper over the top of smear and place the slide over a boiling hot water bath on a mesh surface. • Cover the filter paper with the primary stain, carbol fuchsin. • Leave the slide on the water bath for 5 minutes or more. • Note- Continue to apply stain if the filter paper begins to dry. • Remove the filter paper and rinse the slide with water until the solution runs clear. • Run acid-alcohol decolorizer over the slide for approximately 10 to 15 seconds. • Rinse the slide with water. • Cover the smear with the counterstain, methylene blue, for 1 minute. • Gently rinse the slide with water. • Blot the slide dry with bibulous paper. • Observe under microscope. A.B
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- 11. Observation under Microscope Acidfast: Red, straight or slightly curved rods, occurring singly or in small groups, may appear beaded Non-acid fast: Blue color; In addition, background material stain blue. A.B
- 12. Organisms stained byacid fast staining A.B