AW: [Histonet] RE: Masson Trichrome stain

Sat Mar 14 03:14:29 CDT 2015

Hi Nancy,
saturation of picric acid should be 14 g/l at 20°C  and 60 g/l at 100°C (wikipedia).
Wiki also states 0,04 M for picric acid in Bouins. 229 g/mol --> 9 g picric acid in 1 l Bouin. --> ca. 0,9% (Their Bouin contains 85% saturated picric acid)

recept: 60 ml picric acid, 20 ml formaldehyde, 4 ml acetic acid
Bouin is made of ca. 70% saturated picric acid. My rough estimation is 9,8 g/l picric acid in Bouin. Over the thumb 0,9 % in Bouin.

Romeis states saturated picric acid is 1,2%. That would also bring a 0,9% concentration of picric acid in Bouin.

Another question comes to my mind: We distinguish between hot saturated and cold saturated solution. Is the hot saturated solution after cooling able to hold more picric acid than the cold?

If one starts with 60 g/l for saturated picric acid. This leads to about 3,6%.

I hope mathematics and chemists won't beat me for my calculation. ;-)

Gudrun

-----Ursprüngliche Nachricht-----
Von: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von nancy lowen
Gesendet: Freitag, 13. März 2015 21:57
An: gayle.callis <@t> bresnan.net; histonet <@t> lists.utsouthwestern.edu
Betreff: Re: [Histonet] RE: Masson Trichrome stain

Just out of curiosity--what strength of Picric acid is in your Bouins fixative?Nancy 

     On Thursday, March 12, 2015 2:02 PM, Gayle Callis <gayle.callis <@t> bresnan.net> wrote:

 I have been following the string of inquiries about using metal forceps with Masson's Trichrome staining.  I was taught many years ago to avoid metal forceps or the older metal tissue cassettes with Bouins.  I am scrambling to find the actual reference.  The reason given was acids in Bouins corrode metal.  This may be a lost bit of information since the overall majority of labs now use plastic tissue cassettes.  Case in point:  using acidic descaling solutions for household cleaning i.e. showers/tubs or coffee machines.  These solutions come with warning to avoid metal fixtures and stainless steel sinks.  Accidental contact of acids in a stainless sink causes the metal  discolor, indicating corrosion - been there, done that to a stainless steel sink.  I so use metal forceps to move slides between Mass Tri staining solutions (and silver staining solutions) without problems per John Kiernan's comment.          

Not using metal forceps with silver stains i.e. GMS, reticulin, is to avoid metal ion contamination which is more likely due to with poorly washed glassware.  In the past, we dipped metal forceps in melted paraffin, very messy since paraffin comes off on slides and in hot staining solutions.
Disposable plastic forceps are cheap but break easily resulting in a dropped slide.  Teflon forceps are pricey but it was a challenge to hold slides.
Hopefully there are teflon forceps that work better than the one we used?
We tried a teflon tipped metal forceps but not worth the price as teflon wears off the tips to rexpose metal.    Weigerts hematoxylin  is not affected by metal forceps since there are no acid components to corrode the metal although Weigerts can "stain" the forceps.  Simply wash the forceps in dilute chlorine bleach then soap and water.  I agree with John Kiernan and now use metal forceps to move slides between staining solutions in both Massons trichrome (and silver methods)  without problems.  If people want to use plastic or teflon forceps, I understand the reasons.      

As for not rinsing before going into Aniline Blue (or light green) in Massons trichrome, there is a reason for this.    Sheehan and Hrapchak state verbatim " The phosphomolybdic acid and phosphotungstic acid thus acts as a link connecting basic groups of the connective tissue fiber to the basic groups of the dye i.e. aniline blue.  The PM/PT acid treatment has the ultimate effect of making an amphoteric dye that would ordinarily act as an acid dye to change and act as a basic dye".  These authors also say "Although the exact mechanism of how the stain works is unknown, some theories are available."    By rinsing away the PT/PM, the link may be weaker hence one goes from PT/PM directly into aniline blue (sometimes light green or fast green).    Bierbrich Scarlet/acid fuchsin and aniline blue (light green or fast green) solutions can be filtered back and reused many times.  PT/PM and 1%  acetic acid solutions  should be discarded after use.

Instead of kits due to expense and some kit deviations from classic Massons Trichrome method, I found I could buy excellent, reliable single staining solutions i.e. Biebrich Scarlet/Acid Fuchsin and Aniline Blue from Newcomer Supply or Poly Scientific to avoid exposure when weighing out carcinogenic dyes.  Bouins is purchased from the vendor with the best price.  However, PT/PM and acetic acid single use solutions were still made in house to save costs.      

I strongly recommend reading John Kiernan's  " Methods for Connective Tissues"  from his book , Histological and Histochemical Methods Theory and Practice  for better explanation and understanding of Massons Trichrome chemistry.    Collagen and muscle staining methods in Sheehan and Hrapchaks Theory and Practice of Histotechnology is not recent but a good start.

Whew, a long reply but hope helps...........................

Gayle Callis

HTL/HT/MT(ASCP)  

Written is:  

Justine,

I do not have any metal forceps in the special stains area, due to the reaction that they can cause when staining with silver.  As a rule of thumb, it is just easier to use plastic all the way around.  

The Carson text does not state the use of only plastic forceps, but I would think that maybe they are concerned with a reaction between the Weigert's and the metal.  That would be a stretch.

As for no water before aniline blue, I believe the concentration is very weak and the water may dilute they dye even further.  This would affect the staining results.

Sincerely,

Toysha N. Mayer, D.H.Sc., MBA, HT (ASCP)

Instructor/Education Coordinator

Program in Histotechnology

School of Health Professions

UT M.D. Anderson Cancer Center

713.563-3481

------------------------------

Message: 4

Date: Tue, 10 Mar 2015 00:31:56 -0500

From: John Kiernan <jkiernan
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> uwo.ca>

Subject: Re: [Histonet] FW: Masson's trichrome stain

To: Linda Margraf <lindamargraf
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> gmail.com>,

                histonet
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> lists.utsouthwestern.edu

Cc: justinelanzon
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> hotmail.com

Message-ID: <7380eaed48941.54fe3b7c
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> uwo.ca>

Content-Type: text/plain; charset=iso-8859-1

The notion of plastic forceps is new to me. Where did Justine find it?
Nothing in any variant of the Masson procedure should be adversely affected by moving slides with stainless steel forceps. Is there a commercial campaign to sell plastic tweezers to Histonetters? 

John Kiernan

= = =

On 08/03/15, Linda Margraf  <lindamargraf <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> gmail.com>
wrote:

> Here is a message from Justine...

> 

> From: Justine Lanzon [mailto:justinelanzon
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> hotmail.com] 

> <justinelanzon
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> hotmail.com]>

> Sent: Thursday, March 05, 2015 5:36 AM

> To: lindamargraf
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> gmail.com

> Subject: Masson's trichrome stain

> Hi,

> I am doing a write up on Masson's trichrome stain however I cannot

> answer these two questions:

> - Why are plastic forceps used instead of metal ones to hold the

> stained slide?

> - Why do we not rinse before Alinine blue?

> Can you please help me?

> Many Thanks,

> 

> Justine Lanzon

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