PAS and AFB.ppt
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The document summarizes the PAS staining technique and Ziehl-Neelsen staining technique for acid-fast bacteria. The PAS stain involves oxidizing carbohydrates using periodic acid, then treating with Schiff's reagent to produce a magenta color. The Ziehl-Neelsen stain takes advantage of mycobacteria's lipid-rich cell wall, using a carbol fuchsin solution which is retained after differentiation in acid or alcohol, allowing visualization of acid-fast bacilli. Both techniques are described in detail, outlining the required solutions and step-by-step procedures.
PAS and AFB.ppt
- 1. Principle: The PAS stain is a histochemical reaction in that the periodic acid oxidizes the carbon to carbon bond forming aldehyde which react to the fuchsin- sulfurous acid which form the magenta color. Glucose + Periodic acid - aldehyde (Carbohydrate) Aldehyde + Schiff’s reagent - magenta colored compound
- 2. PAS Technique: - Periodic Acid Solution Periodic acid - 1 gm Distilled water - 200 ml - Schiff Reagent Dissolve 1gm of basic fuchsin in 200 ml of boiling distilled water. Allow the solution to cool 50 °c, and add 2-gm potassium metabisulphite with mixing. Allow to cool to room temperature then add 2 ml conc. Hydrochloric acid, mix, add 2 gm activated charcoal and leave overnight in the dark at room temperature. Filter, the solution should be either clear or pale yellow color. Store in a dark container at 4°c.
- 3. Procedure: o Dewax sections and bring to distilled water. o Treat with periodic acid, 10 mins. o Wash well with several changes of distilled water. o Cover with schiff’s reagent, 10-20 min. o Wash in running tap water, 5-10 min. o Stain nuclei with Harris’s haematoxylin or an iron haematoxylin. (Do not use Ehrlich’s haematoxylin, which will also stain some PAS positive compounds.) o Differentiate in acid-alcohol . o Wash in water, dehydrate in alcohol, clear in xylene and mount as desired.
- 14. Ziehl-Neelsen (ZN) Stain for Mycobacterium Bacillus Mycobacteria are difficult to demonstrate by the Gram technique because they possess a capsule containing a long chain fatty acids that make them hydrophobic. Phenolic acid or heat may be used to reduce the surface tension and increase the porosity.
- 15. PRINCIPLE Mycobacteria tuberculosis (tubercle bacilli) have a lipid-rich cell wall which is capable of taking up strong phenol dye solutions (eg. carbol fuchsin solution) such that the dye is retained upon subsequent differentiation in acid or alcohol ie. they are said to be acid and alcohol fast (AAFB = acid and alcohol fast bacilli).
- 16. Solutions Carbol – fuchsin 1 g basic fuchsin dissolved in 10 ml of absolute alcohol; add 100 ml of 5% aqueous phenol. Mix well and filter before use. Acidified methylene blue 0.25% methylene blue in 1 % acetic alcohol. Malachite green Blue green counterstain used
- 17. Method 1. Deparaffinize and rehydrate through graded alcohols to distilled water. 2. Flood sections with freshly filtered carbol-fuchsin and heat to steaming with intermittent flaming, 15 minutes, or stain in Coplin jar at 56°-60°C, 30 min. 3. Wash well in tap water. 4. Differentiate in 1 % acid alcohol, 10 min. 5. Wash well in tap water. 6. Counterstain in methylene blue solution, 30 seconds. 7. Blot and differentiate by alternate dehydration and rehydration until the background is a delicate pale blue. 8. Dehydrate, clear and mount.
- 18. PROCEDURE FOLLOWED IN OUR HISTOPATHOLOGY LAB
- 26. Wash in water, dehydrate in alcohol, clear in xylene and mount as desired
- 27. Mycobacterium Nocardia Legionella Micdadei Smegma bacilli Rhodococcus Isospora Spermatic head Cryptococcal cyst
- 28. Mycobacterium tuberculosis in lung, Ziehl-Neelsen acid fast stain.
- 30. 1.Deparaffinize in 2 changes of xylene peanut oil ,6 min each 2.Drain slides vertically on paper towel and wash in warm, running tap water for 3 min 3.Stain in carbol fuschin at room temperature for 3 min 4.Wash in warm running tap water for 3 min and drain excess water 5.Decolorize with 5% sulphuric acid and 25% alcohol two changes of 90 seconds each 6.Wash in warm running tap water for 5 min 7.Counterstain in working methylene blue one quick dip 8.Wash in warm running tap water for 5 min 9.Blot sections and dry in 50-55 C oven for 5 min 10.Once dry one quick dip in xylene then mount
- 34. Thank you