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What Kind of Fungus is This?

You know there’s something wrong with the GMS stain when everything turns black.  Time to make new reagents.  After they are all made, it’s right to test the stain with known control tissue.  After the staining was complete, a variety of black components covered my slides.  The GMS stain is a silver stain that fungi “reduce” and then appear black.  Unfortunately other tissues also can turn black and add confusion to the diagnosis of a type of fungus.  After the slides were dried for 24 hours, I took high-resolution pictures of the black substances in my tissues to hopefully get help in fungi identification from you.  Below are 6 different slides with possible fungi.  If anyone knows what any of these pictures could be please comment below.  All comments are welcome.

  1. Muc 1.1 GMS 20x

 

 

 

 

 

 

 

 

 

 

2. Fungus 2 GMS 100x

 

 

 

 

 

 

 

 

 

 

 

 

3.

FUN 6 GMS 100x

 

 

 

 

 

 

 

 

 

 

 

 

4.

Fun 1.1 GMS 100x

 

 

 

 

 

 

 

 

 

 

 

 

5.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FUN GMS 2.1 100x

 

 

 

 

 

 

 

 

 

 

 

 

6. GMS 1.1 100x

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Identification of Fungus

Mycology, the study of fungus is not something I know too much about especially when it comes to histopathology.  I do know that fungus is not something I want propagating in my organs or tissues in serious quantity.  Certain species can wreak havoc on the internal and external systems of the animal bodies.  The identification of these fungi in tissues is visualized by staining with GMS, PAS and Gridley’s.  The stain fungi are then analyzed under the microscope by a pathologist who determines by size, shape and surrounding tissue changes, what the type of fungus is.  This is a very exact science that takes years of practice and studying to determine a possible diagnosis.  Some histologists may be able to guess at the type of fungus on a slide but only guess.  For a histologist must rely on the shape and color of a fungus determined by the stain.  I do this on a consistent basis, trying to guess what kind of affliction the tissue reveals after staining.  For fungus identification, I have been relying on a website called doctorfungus.  Here I can analyze my slides against a good knowledge base and slides to match.  Thus I feel like sharing this resource with you might help.

http://www.doctorfungus.org/thelabor/histopat.php

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PAS Diastase Stain Protocol

If you are going to do a PAS stain then you might as well run a PAS with diastase also.

Safety equipment: Work under a hood with lab coat, gloves, and glasses.

  1. Two sets of slides and 2 control slides are used
  2. De-paraffin slides in xylene (1) —————— 2 minutes (re-use)
  3. De-paraffin slides in xylene (2)—————— 2 minutes (re-use)
  4. Clear slides in 100% alcohol——————— 2 minutes
  5. Clear slides in 100% alcohol——————— 2 minutes
  6. Hydrate slides in 95% alcohol——————- 2 minutes
  7. Hydrate slides in running water—————– 5 minutes
  8. Set 1 (with a control) in 80% alcohol ———— 60 minutes
  9. Set 2 (with a control) in diastase of malt——— 60 minutes
  10. Combine both sets of slides for remaining steps
  11. Wash in running water ————————– 10 minutes
  12. 0.5% periodic acid ——————————- 5 minutes (re-use)
  13. Schiff’s reagent at 37C————————— 15 minutes (re-use)
  14. Wash in running water ————————– 10 minutes
  15. If staining for glycogen, Hematoxylin ———— 20 seconds (re-use)
  16. Dehydrate in 95% alcohol———————— 30 seconds
  17. Dehydrate in 100% alcohol———————- 1 minute
  18. Dehydrate in 100% alcohol———————- 2 minutes
  19. Dehydrate in 100% alcohol———————- 2 minutes
  20. Clear in xylene (3)——————————- 2 minutes (re-use)
  21. Clear in xylene (4)——————————- 5 minutes (re-use)

Results:         Glycogen, mucin, fibrin, or thrombi, colloid droplets, hyaline of arteriosclerosis, hyaline deposits in glomeruli, granular cells in the renal arterioles where preserved, most basement membranes, colloid of pituitary stalks and thyroid, amyloid infiltration may show a positive reaction – Rose to Purplish Red

                        Nuclei – Blue

 

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Grocott’s Methanamine Silver (GMS)

Here is another fungus stain.  Some of the fungi that you might stain with PAS but do not will stain with the GMS.  The silver used must be disposed of properly (not down the sink).  When using gold chloride remember to use plastic forceps, metal forceps will corrode on contact.  All glassware should be chemically cleaned before use to reduce contamination.  Pictures will be coming soon.

Use Control Tissue with known fungi.

Safety equipment: Work under a hood with lab coat, gloves, and glasses.

  1. De-paraffin slides in xylene (1) —————— 2 minutes (re-use)
  2. De-paraffin slides in xylene (2)—————— 2 minutes (re-use)
  3. Clear slides in 100% alcohol——————— 2 minutes
  4. Clear slides in 100% alcohol——————— 2 minutes
  5. Hydrate slides in 95% alcohol——————- 2 minutes
  6. Hydrate slides in running water—————– 5 minutes
  7. Place in 10% chromic acid ———————- 15 minutes (re-use)
  8. Rinse in running water ————————- 1 minute
  9. Place in 1% sodium bisulfate ——————– 1 minute (re-use)
  10. Rinse in running water ————————- 1 minute
  11. Place in working Methanamine silver at 60C—- 60 minutes
  12. Rinse in running water ————————- 1 minute
  13. Place in 0.1% gold chloride ——————— 4 minutes (re-use)
  14. Rinse in running water ———————— 1 minute
  15. Place in 2% sodium thiosulfate —————- 4 minutes (re-use)
  16. Rinse in running water ———————–  1 minute
  17. Place in working light green ——————  30 seconds
  18. Dehydrate in 95% alcohol——————— 30 seconds
  19. Dehydrate in 100% alcohol——————– 1 minute
  20. Dehydrate in 100% alcohol——————– 2 minutes
  21. Dehydrate in 100% alcohol——————– 2 minutes
  22. Clear in xylene (3)—————————- 2 minutes (re-use)
  1. 23.   Clear in xylene (4)——————————– 5 minutes (re-use)

Results:         Fungi – Sharply delineated in Black      

Mucin – Taupe to dark Grey

                       Inner parts of mycelia and hyphae – Old Rose            

Background – Pale Green

 

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