[Histonet] Gomori's Retic Stain

[gayle callis]

We always made sure the ammonium hydroxide was fresh stock, and stored this
in the refrigerator although the later may not be necessary. If near the
expiration date on bottle, NH4OH was replaced.   Also, we made sure the
silver nitrate solution was fresh, and stored the silver nitrate salts in
the refrigerator as directed by the MSDS or as indicated on the bottle.
These were two facts picked up from workshops along the years from experts
lecturing on the stains. Having fresh silver nitrate was also important for
other silver stains e.g. Gomori Methenamine silver for fungus and also Jones
Basement Membrane stain. 

I have seen silver nitrate stored at RT over a long period of time and the
salt looked more powdery compared to storing in the refrigerator where the
salt was more crystalline in nature.  

Gayle Callis
HTL/HT/MT(ASCP)
Bozeman MT   

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Lee & Peggy
Wenk
Sent: Thursday, November 05, 2009 3:08 AM
To: 'Theresa Rohr'; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Gomori's Retic Stain

Students and I just had a similar problem yesterday! There are lots of
reasons why a retic stain could be light, but since you said you followed
the same protocol, I thought it might be what happened to us:

When mixing the silver nitrate with the ammonium hydroxide, the result was a
yellow color, instead of the gunky gray black the occurs when ammonia is
added to the silver solution (which eventually clears when more ammonia is
added). No matter how much ammonia was being added, it stayed yellowish.

The ammonium hydroxide was near the end of the bottle. Since ammonia likes
to outgas, that meant there was very little ammonia left in the "liquid". We
obtained a new bottle of ammonia, and the stain worked fine. 

Chemistry-wise (my understanding), silver nitrate (AgNO3)is soluble in
water. When ammonium hydroxide (NH4OH)is added, silver hydroxide (AgOH) is
made, which is not soluble in water, so becomes the gunky gray-black
precipiate (real scientific terms, I know). When more ammonia is added, the
silver hydroxide is made into a silver diamine hydroxide (Ag(NH3)2OH), also
known as ammoniacal silver. This silver diamine hydroxide is again soluble
in water, which is why the gray-black gunk redissolves.

If your ammonium hydroxide is weak, then not all the silver hydroxide is
converted into silver diamine hydroxide. Therefore, there is not enough
silver diamine hydroxide to bind to the reticulin fibers (= light staining).
And there is still silver hydroxide in the solution (= precipitate on your
tissue/slides).

Peggy Wenk, HTL(ASCP)SLS
Schools of Histotechnology
Beaumont Hospital
Royal Oak, MI 48073

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Theresa Rohr
Sent: Wednesday, November 04, 2009 3:30 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Gomori's Retic Stain

Histonetters.

Suddenly having a problem with Retic stain.  Fibers weakly staining and dark
silver spots deposited all over slide. 

We seem to be following same protocol we always used but I am wondering if
something I am not noticing has changed.  Could it be too much Ammonium
Hydroxide clearing the precipitate or too little used. Reagents made up new
of course and extra care with acid glass cleaning.  Just thought maybe
someone else had a similar problem and found an easy solution. 

If you have any thoughts I would be very appreciative.  

Theresa Rohr, BA, HT
Nyack Hospital, NY
rohrt <@t> nyackhospital.org

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