Re: [Histonet] Grocott's Methenamine Silver Stain

From:Jackie.O'Connor@abbott.com


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                                                            
                       ,                              
  Sent by:                "'histonet@lists.utsouthwestern.edu'"               
  histonet-bounces@lists.                  
  utsouthwestern.edu              cc:                                         
                                  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-
Kathleen

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.
>>
>>Thanks,
>>Kathleen
>>
>>Wayne Holland wrote:
>>
>>
>>
>>>Make sure your solutions are fresh, especially the silver. ALso try
>>>10% chromic for 10 minutes.
>>>
>>>
>>>
>>>
>>>>From: Kathleen Roberts 
>>>>To: "'histonet@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
>>>>hour).
>>>>
>>>>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
>>>>
>>>>
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>>>>
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>>
>>
>>
>Gayle Callis
>MT,HT,HTL(ASCP)
>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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