[Histonet] Grocott's Methenamine Silver Stain

Jackie.O'Connor <@t> abbott.com Jackie.O'Connor <@t> abbott.com
Wed May 5 15:03:13 CDT 2004

In an attempt to remove hazardous chromic acid from the lab, I vaguely
remember replacing the Chromic Acid with 5% Periodic acid instead for the GMS.
Has anyone else done this?  If my memory serves me correctly - it works fine -
but I am old  . . . . er.  (must be from all the teenagers at home).

Jacqueline M. O'Connor HT(ASCP)
Abbott Laboratories
Global Pharmaceutical Research and Development
Discovery Chemotherapeutics

  Kathleen Roberts                                                            
  <kgrobert <@t> rci.rutgers.e         To:        Gayle Callis                     
  du>                     <gcallis <@t> montana.edu>,                              
  Sent by:                "'histonet <@t> lists.utsouthwestern.edu'"               
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                                  Subject:        Re: [Histonet] Grocott's    
                          Methenamine Silver Stain                            
  05/05/2004 02:58 PM                                                         

One thing-this wasn't a kit that I used, and I *am* following the
protocol in the AFIP manual.  The chromic acid is a 10% solution from
LabChem, and I diluted it down to 4%.  I suppose that they could have
made a mistake in making the solution.

How do you make up your chromic acid?

Thanks for your help-

Gayle Callis wrote:

>I don't thing 10% chromic is the answer, the method calls for 4%.  Maybe
>they made a mistake making it? Is it actually stronger than they say?
>You don't want to have Chromic too concentrated as if you overoxidize you
>will take the vicinal OH groups to aldehyde and then beyond and get zip for
>results.  I have never used ready to use kits, and make up my 4% chromic
>acid fresh each time. It is possible to OVEROXIDIZE with this method.
>A good read on this method is AFIP manual, Lee Luna tells HOW it should be
>done very clearly.
>At 03:18 PM 5/5/2004 -0400, you wrote:
>>I made up everything fresh just before I did the stain... :oP    I'll
>>try the 10% chromic acid.
>>Wayne Holland wrote:
>>>Make sure your solutions are fresh, especially the silver. ALso try
>>>10% chromic for 10 minutes.
>>>>From: Kathleen Roberts <kgrobert <@t> rci.rutgers.edu>
>>>>To: "'histonet <@t> lists.utsouthwestern.edu'"
>>>><histonet <@t> lists.utsouthwestern.edu>
>>>>Subject: [Histonet] Grocott's Methenamine Silver Stain
>>>>Date: Wed, 05 May 2004 13:54:18 -0400
>>>>Hey, all-
>>>>I've been trying to stain some FFPE slides of decalcified rat sinuses
>>>>for fungi using the AFIP protocol for Grocott's stain, and despite
>>>>using fresh reagents, the positive control and unknowns do not stain
>>>>at all, except for some random deposition of silver that does not
>>>>even begin to look like fungi.  The positive control is loaded with
>>>>Candida, and can be seen with H&E easily.
>>>>So far, we don't see any fungi in the rat sinuses and lungs (they
>>>>were sneezing, and the bedding came back positive for Aspergillus)
>>>>with H&E staining, which is why we decided to try the Grocott's-one
>>>>way or another we should be able to visualize it in the tissues.
>>>>Any ideas why the Candida-loaded positive control would not stain
>>>>with Grocott's?  Right now I am thinking that perhaps they didn't sit
>>>>long enough in the 4% chromic acid, even though the protocol says to
>>>>soak the slides for an hour to oxidize them. Either that or a longer
>>>>soak in the methenamine-silver solution at 60 degrees C (also for one
>>>>Thanks in advance for your help-
>>>>Kathleen Roberts
>>>>Principal Lab Technician
>>>>Neurotoxicology Labs
>>>>Dept of Pharmacology & Toxicology
>>>>Ernest Mario School of Pharmacy
>>>>Rutgers University
>>>>41 B Gordon Rd
>>>>Piscataway, NJ 08854
>>>>Histonet mailing list
>>>>Histonet <@t> lists.utsouthwestern.edu
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>Gayle Callis
>Research Histopathology Supervisor
>Veterinary Molecular Biology
>Montana State University - Bozeman
>PO Box 173610
>Bozeman MT 59717-3610
>406 994-6367 (lab with voice mail)
>406 994-4303 (FAX)

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