[Histonet] Chromic vs Periodic Acid to oxidize tissues, and use of Thiosemicarbazide, for Grocott's Methenamine Silver (GMS) for fungi?

[Hood, Jordan]

Hi all,

A while back I asked you about how to successfully perform Jones' Methenamine Silver (JMS) stain on formalin-fixed paraffin-embedded pig kidneys, and your suggestions to use Thiosemicarbazide (TSC) after oxidation with Periodic Acid solved my issue with the silver solution instability (since the slides didn't have to sit in there for too long) and it yielded great results. I have now been asked to generate a protocol (for my research histology/pathology lab specializing in animal tissues) for Grocott's Methenamine Silver (GMS) for Fungi, and while I plan on more-or-less following the protocol from Rowley Biochemical (https://rowleybiochem.wpengine.com/wp-content/uploads/A-121-Grocotts-Method-GMS-for-Fungi.pdf), I have a couple of questions for you:


  1.  Will TSC work after oxidation with Chromic Acid (Rowley says to use Chromic Acid)?
  2.  If TSC won't work after Chromic Acid, can I oxidize using Periodic Acid and then use TSC as I did for JMS? I found one article/abstract (https://pubmed.ncbi.nlm.nih.gov/25861133/) that says that it's possible to use heated Periodic Acid, but most other sources indicate that I should use Chromic Acid.
  3.  Can I use the same silver solution reagents and mixing procedure as I did in JMS (I already have these in the lab and already know that they work, so ideally I wouldn't have to deviate from my JMS protocol)?
     *   For JMS (mostly taken from Rowley's protocol), I made up and filtered the silver solution immediately before use by mixing:

                                                               i.      170 ml Methenamine 3% (Rowley; F-396-2)

                                                             ii.      10 ml Silver Nitrate, 5% (Rowley; F-396-3) and

                                                           iii.      48 ml Borate Buffer, pH 8.2 (Rowley; F-396-4).

     *   Rowley says that for GMS, I should:

                                                               i.      First, prepare Methenamine-Silver Nitrate Stock:

           *   Silver Nitrate, 5%, Aq. (Rowley; A-121-3A) - 5ml
           *   Methenamine, 3%, Aq. (Rowley; A-121-3B) - 100ml

                                                             ii.      Use chemically clean glassware. Store in refrigerator.

                                                           iii.      Then, to prepare Methenamine-Silver Nitrate Working, mix just prior to use:

           *   Methenamine Silver Nitrate Stock - 25.0ml
           *   Distilled Water - 25.0ml
           *   Sodium Borate, 5%, Aq. (Rowley; A-121-2) - 2.0ml

I am certainly open to any general advice that you have for GMS as well.

Thank you!

Jordan H.
University of Michigan
Ann Arbor, MI
**********************************************************
Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues