Periodic acid Schiff’s (PAS) Stain: Introduction, Principle, Procedure, Result Interpretation

Periodic acid Schiff’s (PAS) Stain: Introduction, Principle, Procedure, Result Interpretation

Introduction of Periodic acid Schiff’s (PAS) Stain

Periodic acid Schiff’s (PAS) stain is used for the demonstration of basal membrane, fungus, differentiate mucin secreting adenocarcinoma from undifferentiated squamous cell carcinoma. It is helpful in the diagnosis of different types of glycogen storage diseases, Paget’disease, macrophages in Whipples diseases, parasites, and amylase.

Principle of Periodic acid Schiff’s (PAS) Stain

Structures having 1-2 glycol or amino or alkyl grouping are oxidized by periodic acid. Free hydroxyl groups must be present for oxidation. After complete oxidation, a dialdehyde is formed which is colorless and unstable. This aldehyde after treating with Schiff’s reagent turned to a magenta red-colored product.

Test Requirements for Periodic acid Schiff’s (PAS) Stain

Reagent

  • Hematoxylin
  • Periodic acid
  • Hydrochloric acid (HCl)
  • Potassium
  • Meta bisulfate
  • Alcohol
  • Activated charcoal
  • Basic fuchsin

supplies

  • Gloves
  • Masks
  • Lab coat/ apron
  • Cotton

Equipment

  • Coplin jar
  • Measuring cylinder
  • Conical flasks
  • Glassware
  • Reagents bottles

Preparation and Procedure of PAS stain

Preparation of reagents (0.5% Periodic acid)

  • Periodic acid: 0.5 gm
  • Distilled water: 100 ml

Preparation of Schiff reagent

  • basic fuchsin – 1 gm
  • distilled water – 200 ml
  • potassium meta bisulfate – 2 gm
  • Concentrated HCl – 2 ml
  • activated charcoal – 2 gm
  1. Dissolve 1 gm of basic Fuschia in 200 ml of boiling distilled water by removing the flask of water from the burner just before adding the basic fuchsin.
  2. Allow the solution to cool at 50°C.
  3. Add 2 gm of potassium metabisulphite and mix properly. Allow cooling to room temperature.
  4. Add 2 ml concentrated hydrochloric acid and mix.
  5. Leave overnight in the darkroom temperature and add 2 gm activated charcoal. Filter through a no. 1 Whatman filter paper, the solution should be
    colorless.
  6. Store in an amber-colored container at 4°C.

Preparation of Harris’s Hematoxylin ( 500ml )

  • Hematoxylin – 2.5 g
  • Absolute alcohol – 25 ml
  • Potassium alum – 50 gm
  • Distilled water – 500 ml
  • Mercuric oxide – 1.25 g
  • Glacial acetic acid – 20 ml

 

  1. Dissolved Hematoxylin in absolute alcohol
  2. Take hot distilled water and mix alum until dissolved well
  3. Add mercuric oxide carefully and cool in cold water, taking care of
    excess bubbling.
  4. Add glacial acetic acid

Procedure of Periodic acid Schiff’s (PAS) Stain

  1. Deparaffinize and hydrate to distilled water.
  2. Place slide into 0.5% periodic acid for 5-7 minutes.
  3. Wash with several changes of distilled water.
  4. Cover with Schiff’s solution for 20 minutes until dip magenta color is seen.
  5. Then wash in running tap water.
  6. Counterstain with Harris hematoxylin for 1 minute.
  7. Wash in running tap water.
  8. Dehydrate in alcohol, clear in xylene, and mount in D.P.X.

Result interpretation of Periodic acid Schiff’s (PAS) Stain

Nuclei: Blue
Glycogen, fungus: Magenta/ deep pink
Diastase treated tissues: Colorless

Note: Fungi stain a bright pink-magenta or purple against a green background when light green is used as a counterstain.

Precautions for Mycological Aspects

A slide of either skin or nail scrapings containing a dermatophyte should be stained along with slides of the specimen as a positive control. Periodic acid may deteriorate and no longer oxidize the hydroxyl groups. This should be suspected when fungal elements on the control slide appear unstained. The
periodic acid solution and the stock of periodic acid (a white powder)
should be kept in dark bottles. The sodium metabisulphite solution is unstable. Deterioration of this reagent is suspected when the control slides
show no evidence of having been subjected to a bleaching process, e.g. the
background stains as intensely as they do the fungal elements.

Further Readings

  1. Bancroft’s  Theory and Practice of Histological Techniques(6 th edition)
  2. .Topley and Wilson’s Microbiology and Microbial Infections. Volume 4, Tenth
    Edition, 2005.
  3. Rippon JH. Medical Mycology. The pathogenic fungi and the pathogenic
    Actinomycetes. Third Edition, WB Saunders Company, 1988.
  4. A Textbook of Medical Mycology. Editor: Jagdish Chander.  Publication Mehata, India.
  5.  Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore
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