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Histotechnology and Diagnostic Cytology

WHAT IS NEW WITH THE PAS STAIN?

There have been several interesting developments in the performance of the old established Periodic Acid-Schiff reaction for carbohydrate and mucosubstances and I would like to summarise them.

Efforts have been made to simplify the preparation of the Schiff reagent. Horobin and Kevill-Davis (Stain Techn 46:53-58, 1971) described an alcoholic Schiff reagent with the following formulae:

• Basic Fuchsin 0.5g
• Ethanol 80ml
• Distilled Water 20ml
• Hydrochloric acid 1ml

After 20 minutes staining, excess dye is removed with alcohol.

Since the activity of periodic acid in alcohol is unaffected, cytology may find this modification especially useful for the demonstration of glycogen and mucins that are particularly water soluble.

Garvey etal (J.Histochem 15(2):117-120, 1992) developed a modified Schiff solution containing pararosaniline hydrochloride in a concentration of about 1/10 of that normally used for basic fuchsin. They doubled the concentration of sodium hydrosulphite to decolourise the pararosaniline more effectively. Their formulae is as follows:

• Pararosaniline Hydrochloride (CI 42500) 0.15g
• 20% ethanol 10ml
• Dissolve with the aid of heat
• Distilled water 90ml
• Sodium Metabisulphite 2g
• Sodium Hydrosulphite 0.1g
• Stir well
• Concentrated Hydrochloric acid 1ml
• Add 1g activated charcoal, shake well and filter

Perera (J. Histotech., 13(3):155-158, 1991) have described a method where a dilute solution of basic fuchsin is mixed with a diluted solution of periodic acid and the mixture is used to demonstrate PAS positive substances. Their technique follows:

1. Dewax and hydrate sections.
2. Flood sections with freshly prepared PABF mixture and stain for 5-8 minutes:

• 0.2% Basic Fuchsin 2ml
• 1% Periodic Acid 4ml

1. Wash slides in water.
2. Differentiate in 0.5% acid alcohol until no more red colour leaves the section.
3. Wash in tap water and dip slide in 1% ammonia alcohol
4. Counterstain as desired, dehydrate clear and mount.

A useful quality control procedure for formalin concentration has been described by Jaspers (J. Histotechnol., 10(4):263-266, 1978). It can be used to titrate a solution of buffered formalin whose concentration is not known:

1. Take 10ml sample of buffered formalin.
2. Add 50ml of 2.4% Sodium Bisulphite and allow to react for 15min at room temperature.
3. Add 1ml Schiff's reagent

If the solution turns deep violet, the formalin is usable for fixation (ie in excess of 4%).

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