Re: [Histonet] Grocott's Methenamine Silver Stain

From:Kathleen Roberts

As for the decal, I was afraid that the decal solution (Fisher's Cal-EX) 
might have something to do with it...but the control block was tissue 
that did not need to be decal'd, so I think something else is afoot, and 
the decal isn't the whole story.

True, about there possibly being no fungi in the lungs...these rats were 
sneezing, not coughing or wheezing, so I am told, so it may not have had 
time to spread to the lungs, but the vet wanted to check.  H&E of some 
of the lungs does show an inflammatory reaction, however, even though 
there appear to be no fungi.  Little to no inflammation seen in the 
sinuses, though that may have been eaten away by the decal, possibly.

As for waterbath vs. incubator....I used an incubator.  I'll try the 
waterbath next time.

And I agree with you about comparing Candida control with 
Aspergillus...but this block is all we have right now, and the fact that 
*nothing is staining at all* with Grocott's, neither Candida control nor 
Aspergillus-infected subjects is fishy.

Mmm, should I preheat the methenamine silver solution to 60 degrees C 
before putting the slides in?  My protocol didn't say to do that, but I 
think someone here on the list said they did that...must search the 

Thanks for your input!

Gayle Callis wrote:

>It may be that your decalcification (if you used a strong acid and not
>controlled with endpoint determination) may have messed up the stain.  
>EDTA may have been a better choice for decalcification. However, you don't
>decalcify lungs, and if you can't see the fungus in lung, Aspergillus is so
>easy to stain with GMS, then there may not be any fungus there. Candida is
>always a snap to stain, 1 hour in FRESH 4% chromic acid is standard.   GMS
>with Candida and Aspergillus usually comes up within or before 1 hour.  You
>should use a microscope to control silver deposition in positive Candida
>staining.  Are you using a waterbath or incubator?  Waterbath is far better
>for temperature control, more even distribution of heat, incubators have
>convectional air currents.  To check staining, dip in cold water, examine
>then dip in hot distilled water to equilibrate section back to methenamine
>silver temperature.  If you don't dip in cold water, you get steam on your
>microscope objectives. Look for that dark golden brown color at around 30
>min before pulling slide, then check every 15 minutes. 
>Caveat: beware comparing positive Candida staining to your Aspergillus as
>these ARE different fungi, Candida IS different and really stains readily.
>Aspergillus may still be in yeast form phase of infection and not have
>mycelium (a = plural) formation at point you euthanized rat.  Seeing
>Aspergillus readily in H&E is not always the case as with a heavy infection
>of Candida, so be careful how you interpret these two in same breath. 
>Good luck    
>At 01:54 PM 5/5/2004 -0400, you wrote:
>>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
>>Histonet mailing list
>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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