[Histonet] Sandersons Rapid Bone stain chemistry and history

Gayle Callis gayle.callis at bresnan.net
Tue Sep 27 12:19:17 CDT 2016

You worte:  RBS is the same as Methylene Blue, I believe.


Jessica Riggleman | Research Associate


Globus Medical, Inc.

Valley Forge Business Center

2560 General Armistead Avenue | Audubon, PA 19403

Ph: (610) 930-1800 ext. 2583 | Fax:



-----Original Message-----

From: Alicia Marie Ortega [mailto:alicia.ortega at colorado.edu
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> ]

Sent: Thursday, September 22, 2016 4:50 PM

To: histonet at lists.utsouthwestern.edu

Subject: [Histonet] Question on Sanderson Rapid Bone Stain


Hello everyone,


I am attempting to stain 30-40 micron thick undecalcified bone sections
embedded in poly(methyl methacrylate) with Sanderson Rapid Bone Stain (RBS)
with a Van Geison counter stain.

My first attempt at this stain resulted in a very faint stain from the RBS
(I could see very faint blue/green staining of osteoid/soft tissues) and a
very intense dark pink stain from the counterstain.  I was wondering if
anyone knows of a way to intensify the staining of the RBS?  I heated the
stain to 55-60 degrees Celsius as recommended by the manufacturer prior to
staining (with a 10 minute stain duration).

Thank you in advance for your time and help.


Alicia Ortega

Postdoctoral Research Associate

University of Colorado, Boulder



Sandersons Rapid Bone Stain is oxidized methylene blue.  It is actually a
modified  Stevenels blue, a  polychromatic stain made by oxidizing methylene
blue with potassium permanganate that produces the byproducts i.e. methylene
violet, azure A, azure B, thionin, toluidine blue, thionin and possibly some
other thiazine dyes .Stevenels blue was first used by Maniatopoulos et al to
stain PMMA embedded, un-etched bone sections using a 60°C water bath and
optional counterstaining with either Van Gieson or basic fuchsin.   Cathy
Sanderson (Mayton) found a better,  easier way to make up Stevenels blue for
large production.   We found it tends to be a weaker stain unless you etch
the bone with 0.5% formic acid using a sonicator for 1 minute, rinse with
hot tap water very briefly, blot and look at the surface stained bone.
Over aggressive water rinsing allows the stain to release from this mildly
acid etched calcified bone.  All the etching is doing is a gentle removal of
calcium from only a few micrometers of bone surface and allow the stain to
penetrate better.   Acid etching intensified RBS staining but there are
drawbacks but ways to deal with that.   You can stain etched bone sections,
view, photograph and then do a very brief (only a few quick dips), blot
after counterstain, view and photograph the now counterstained section.
Avoiding over rinsing keeps the RBS in etched bone section.   If things
don’t look right, merely polish the stain from surface and start over.   


You can counterstain Sanderson RBS stained (etched) bone section with the
two mentioned counterstains, but it has to be done very briefly or the
counterstain will differentiate RBS from the bone and be overly red.
RBS, as used according to Sanderson and product brochure instructions, is
for un-etched mineralize bone sections in PMMA or even EXAKT preparations.
We preferred to acid etch, do the original Stevenels Blue instead of RBS,
and get a much deeper stain on osteoid, and other bone components.
Counterstaining was not done as it masked acid etched bone components
excessively or removed them,  

The joy of using RBS is avoiding a long, tedious, and messy making up of
Stevenels blue.   One can also try to stain longer in RBS per brochure
instructions to see if that deepens the staining.   Old bone picks up the
stain differently than young bone in terms of age of the animal.  Different
species can stain differently too.  


It is important to know that repeated heating of RBS (or Stevenels)  allows
the portassium permanganate to continue oxidizing the methylene blue, and
also keeps raising the pH  to be more alkaline.  One should replenish or top
off  the stain, always filter it into a clean coplin jar before using or
reusing this stain.  Eventually the stain does not work very well and you
need to start with new stock.  


We preferred to use a MacNeals tetrachrome combined with toluidine blue per
Sterchi method for more brilliant staining of osteoid other bone components
including cartilage.   


Take care


Gayle Callis


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