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Grocott Hexamine-Silver Special Stain For Fungus – Method and Tips


The Grocott Hexamine-Silver special stain is the method of choice for a large majority of histopathology laboratories for the demonstration of all fungi. The formalin-fixed sections are exposed to chromic acid which reacts with fungal cell wall polysaccharide components to form chromic acid-aldehydes. These then reduced by a hexamine-silver solution at an alkaline pH. This causes them to be selectively blackened.  It should be noted that this method is not specific for fungi but rarely fails to demonstrate any fungi within the test tissue.

Below is the author’s method of choice.

Solutions

5% aqueous chromic acid (chromium trioxide)

1% aqueous sodium metabisulphite

Stock hexamine-silver solution = 100ml 3% aqueous hexamine + 5% aqueous silver nitrate.

Working hexamine-silver solution = 2ml 5% aqueous sodium tetraborate (borax) + 25ml distilled water. Mix, then add 25ml stock hexamine-silver solution.

0.1% aqueous gold chloride

5% sodium thiosulphate

0.2% light green in 0.2% acetic acid

Method

1. Take sections to water.

2. Treat sections with chromic acid for 1 hour.

3. Wash thoroughly in tap water.

4. Treat with sodium metabisulphite solution for 1 minute.

5. Wash well in tap water.

6. Wash well in several changes of distilled water.

7. Treat sections with working hexamine solution (preheated in coplin jar at 56 degrees Celsius) at 56 degrees Celsius for 10-20 minutes. Check control sections to see if fungi are a dark brown colour, if not return to solution checking regularly at 3 minute intervals until correct colour achieved.

8. Wash in several changes of distilled water.

9. Treat sections with 0.1% aqueous gold chloride for 3 minutes.

10. Wash well in distilled water.

11. Treat sections with 5% sodium thiosulphate for 5 minutes.

12. Wash well with tap water.

13. Counterstain with 0.2% light green in 0.2% acetic acid for 1 minute.

14. Wash well in tap water.

15. Dehydrate, clear and mount.

 

Tips

– As with all other silver stains wash everything that you are going to use thoroughly with distilled water.

– Store the stock hexamine-silver solution at 4 degrees Celsius away from sunlight. It will keep for 1-2 months. If a white precipitate forms give it a good shake and it should redissolve.

– Do not extend the time in chromic acid too long as this can over oxidize the carbohydrates to carboxylic acid and therefore not take up the silver stain.

– Do not reduce the time in chromic acid as this will lead to under oxidation and therefore no take up of the silver stain.

– The chromic acid can be reused but its efficiency will decrease after each use.

– If the control sections are failing to stain even after extending the staining time in the heated hexamine-silver solution you have more than likely forgotten to add the borax. If so, you can just add it and continue with the stain.

– If you have a large amount of silver precipitate over your sections it is probably due to using low-grade silver.

– If you forget the sodium thiosulphate step you will not realise until after retrieving the slide from storage further in the future, as the remaining silver not removed will react with sunlight turning black.

– Try to keep your counterstain fairly light as dark counterstaining can mask fungal elements.

 

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Periodic-Acid Schiff Diastase (PASD) Special Stain – Method and Tips


In my previous post I covered the Periodic Acid-Schiff reaction (PAS) special stain which is by far the most common stain performed in a routine histology laboratory. A variation on this technique call the Periodic Acid-Schiff Reaction with diastase digestion (PASD) is another commonly performed special stain which I will be covering in this post.

The variation on the PAS technique involves simply exposing the section to the diastase enzyme amylase prior to continuing with the standard PAS method. The term ‘diastase’ refers to any enzyme that catalyses the breakdown of starch into maltose the dextrose. The diastase enzyme acts by cleaving the a-glucosidic 1-4 linkages of starch or glycogen (aka animal starch) leading to the formation of maltose and dextrose (maltose and dextrose are water-soluble sugars). So when sections are pre-exposed to diastase before commencing the PAS technique the glycogen within the tissue is broken down into maltose and dextrose which are dissolved and washed away when the section is rinsed sufficiently in tap water.

The diagnostic purpose of performing the PASD technique include

–         the removal of glycogen to make it easier to identify mucins stained by the PAS technique

–         analysis of glycogen deposits within the liver

–         highlighting a thickened basement membrane for example in lupus

Solutions

Diastase solution – 1 part human saliva to 9 parts distilled water.

1% aqueous periodic acid – from PAS method.

Schiffs Reagent – from PAS method.

 

Method

 

1. Take sections to water.

2. Expose sections to diastase solution for 30 minutes at room temperature.

3. Wash sections thoroughly in tap water

3. Continue with PAS method from step 2

Tips

– Always run a PAS and a PASD control with every batch of PASD stains to ensure your diastase solution is working. This author has found a glycogen rich liver control to be most sufficient. This author also runs a PAS and PASD for all PASD requests.

– Commercial amylase is available instead of using a saliva solution. Commerical amylase sometimes requires different incubation temperatures and conditions so check this before using. This author has found that a saliva solution is easiest due to its ease of preparation, availability and plus it is free.

– Put all sections onto to ‘sticky’ slides ie. Superfrost plus slides or their equivalent as the saliva solution causing some lifting of the section from the slide. This is reportedly more prevalent in sections exposed to the commercial amylase solution.

Thanks for reading and I welcome any comments.

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Keep an eye out for my website (www.skinpathonline.com) COMING SOON.

 

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