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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

 

An Expert Histology Service

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Periodic Acid Schiff (PAS) Stain Protocol

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. 0.5% periodic acid ——————————– 5 minutes (re-use)
  8. Schiff’s reagent at 37C—————————- 15 minutes (re-use)
  9. Wash in running water ————————— 10 minutes
  10. If staining for glycogen, Hematoxylin ——— 20 seconds (re-use)
  11. If staining for fungus, light green ————— 30 seconds (re-use)
  12. Dehydrate in 95% alcohol———————— 30 seconds
  13. Dehydrate in 100% alcohol———————- 1 minute
  14. Dehydrate in 100% alcohol———————- 2 minutes
  15. Dehydrate in 100% alcohol———————- 2 minutes
  16. Clear in xylene (3)——————————— 2 minutes (re-use)
  17. Clear in xylene (4)——————————— 5 minutes (re-use)

Results:           Hematoxylin – 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

Results:          Light Green – Fungi – Red                 Background – Pale Green

 

 

An Expert Histology Service

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Special stains protocols

I am trying to figure out what to do about the special stains protocols.  There are about 35 of them.  Putting them on the front page does not seem to be efficient.   I would like them all to be searchable and downloadable, maybe on a different page.  What do you think, does it make a difference?