Introduction of Periodic acid Schiff’s (PAS) Stain
PAS stain is used for the demonstration of basal membrane, fungus, differentiate mucin secreting adenocarcinoma from undifferentiated squamous cell carcinoma. It is helpful in diagnosis of different type of glycogen storage diseases, Paget’disease, macrophages in Whipples diseases, parasites and amylase.
Principle of 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 magenta red colored product.
Hydrochloric acid (HCl)
Lab coat/ apron
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 metabisulphate – 2 gm
- Concentrated HCl – 2 ml
- activated charcoal – 2 gm
- Dissolve 1 gm of basic fuschin in 200 ml of boiling distilled water by removing the flask of water from the burner just before adding the basic fuchsin.
- Allow the solution to cool at 50°C.
- Add 2 gm of potassium metabisulphite and mix properly. Allow to cool to room temperature.
- Add 2 ml concentrated hydrochloric acid and mix.
- Leave over night in the dark room temperature and add 2 gm activated
charcoal. Filter through a no. 1 whatman filter paper, the solution should be
- Store in 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
- Dissolved Hematoxylin in absolute alcohol
- Take hot distilled water and mix alum until dissolved well
- Add mercuric oxide carefully and cool in cold water, taking care of
- Add glacial acetic acid
Procedure of PAS stain
- Deparaffinize and hydrate to distilled water.
- Place slide into 0.5% periodic acid for 5-7 minutes.
- Wash with several changes of distilled water.
- Cover with Schiff’s solution for 20 minutes until dip magneta color seen.
- Then wash in running tap water.
- Counter stain with Harris hematoxylin for 1 minute.
- Wash in running tap water.
- Dehydrate in alcohol, clear in xylene and mount in D.P.X.
Result interpretation of 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 counter stain.
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 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 the do the fungal elements.
- Bancroft’s Theory and Practice of Histological Techniques(6 th edition)
- .Topley and Wilson’s Microbiology and Microbial Infections. Volume 4, Tenth
- Rippon JH. Medical Mycology. The pathogenic fungi and the pathogenic
Actinomycetes. Third Edition, WB Saunders Company, 1988.
- A Text Book of Medical Mycology. Editor: Jagdish Chandar. Publication Mehata, India.
- Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore