[Histonet] RE: Masson Trichrome stain

Fri Mar 13 15:56:42 CDT 2015

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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