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Hygroscopic Chemicals Cause Stress

Last year I had problems with the trichrome stain. The nuclei would not stain that dark purple back color. I tried making new chemicals one after the other. They were not out of code but maybe someone had poured one into the other or it had become diluted by excess carry over or water. In between each chemical change, the stain had to be tested and quality assessed, a tedious process. After all 7 chemicals had been replaced, the nuclei still appeared washed out.
What could it be, the hematoxylin? I ran the tissues in Weigert’s hematoxylin for the range of 10, 20 and 30 minutes. The nuclei were a little darker at 30 minutes but not enough to get happy over.
After months of trial and error, mostly error, I somehow stumbled upon a clue. During my search of the internet and really just grasping for any hint pointing to anything, I began reading the MSDS of all the chemicals. Let me tell you how to fall asleep even when you’re not to, read the MSDS of all the chemicals used by histology labs. There was a word “hygroscopic” that I was not familiar with. My definition taken from reading many others, a chemical that absorbs water from the atmosphere unless protected against. The Wikipedia definition is:

Masson's TrichromeOne of the recent questions on histonet was “Recently we started having problems with our 10% nb formalin not completely fixing our animal tissues. We haven’t done anything different in making up the solutions, but we’re finding incomplete fixation in the center of the tissues. We are letting it stay in formalin the same amount of time (24-48 hrs.) as we’ve always done. The pH has not changed. Does anyone have any suggestions as to where we can look to troubleshoot correcting this problem? Our recipe method is: 4g Sodium Phosphate, monobasic monohydrate (Fisher Chemical co.) 6.5g Sodium Phosphate, dibasic anhydrous (EMD Chemical co.) 37% Formaldehyde (EMD 10 parts formaldehyde to 90 parts Dh20. Any helpful thoughts would be appreciated.”

My response was as follows:
Hello Fran, I did not see any other replies to your question and hope I am not the
only one. Your recipe looks good and the only thing I notice is the
Sodium Phosphate, dibasic anhydrous. This chemical is hygroscopic,
meaning it takes on water very easily, even out of the air. When this
happens, your intended solution concentration is skewed and may not
do as intended. All of our chemicals that have this property are either stored in
glass desiccate jars in the refrigerator or wrapped with parafilm
after each use and stored in a desiccate jar at room temp.

Update:  We now store all our hygroscopic chemicals in the refrigerator at 4C.

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Masson’s Trichrome Blue Protocol

Masson’ Trichrome Blue
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. Bouins at 60 degrees Celsius————————– 1 hour (re-use)
8. Wash in running water ——————————– 10 minutes
9. Weigert’s Hematoxylin (make fresh) —————— 10 minutes (dump)
10. Wash in running water ——————————– 10 minutes
11. Biebrich Scarlet Acid Fuchsin————————- 10 minutes (re-use)
12. Rinse in running water——————————– 2 minutes
13. Phosphomolybdic / phosphotungstic acid—————- 10 minutes (re-use)
14. No rinse, goes directly into blue
15. Analine Blue—————————————— 10 minutes (re-use)
16. Rinse in running water——————————– 1-2 minutes
17. 1% Glacial Acetic Acid——————————– 5 minutes (re-use)
18. Dehydrate in 95% alcohol—————————— 1 minute
19. Dehydrate in 95% alcohol—————————— 1 minute
20. Dehydrate in 100% alcohol—————————– 1 minute
21. Dehydrate in 100% alcohol—————————– 2 minutes
22. Dehydrate in 100% alcohol—————————– 2 minutes
23. Clear in xylene (3)———————————– 2 minutes (re-use)
24. Clear in xylene (4)———————————– 5 minutes (re-use)

Nuclei- Black
Cytoplasm, keratin, muscle fiber and intercellular fibers- Red
Collagen- Blue