PMMA embedding mixtures

<< Previous Message | Next Message >>
From:Gayle Callis <> (by way of histonet)
To:histonet <>
Content-Type:text/plain; charset="us-ascii"

Diane Sterchi's method used specifically for doing undecalcified
bone microtomed sections or ground sections for surface staining,
large implants or larger bone sections that cannot be microtomed.

Her mixture is (and the one I use exclusively)

1.5 gms benzoyl peroxide(BPO) 99% pure, water free from Aldrich
95 mls mma monomer (Fisher)
10 gms polymethylmethacrylate beads (Aldrich, 996,000 molecular weight)
5 mls dibutyl phthlate

the dibutyl phthlate can be varied to provide harder plastic (use less to give
a harder plastic, does not bend or flex with less of the plasticizer in the
mixture, hence her variables of 1.5 mls to 5 mls)  When the amount is
5 mls, you get a plastic that cuts very well with the Tungsten carbide
blade on a microtome)

BPO is the catalyst.  No accelerator is added, to start the polymerization
with her method (nn dimethylaniline in Jacksons, as is used in GMA), since
you are adding prepolymerized beads to the mixture, polymerization will
occur, but more slowly than when adding the nnDMA).  Her method also
has the advantage of never having to "wash" or remove the inhibitor
(hydroquinone) added by the manufacturer.  The reason so much BPO is
added to some mixtures is to compensate and overcome the inhibitor in
order for the MMA to polymerize.  The inhibitor is the reason monomer
is washed with a sodium hydroxide solution.  It either has to be
removed or overcome for successful polymerization, sorry for the repeat!

Neil Hand has a method similar to Dianes, for glass knife, thin section
microtoming then doing some elegant IHC, Peter Jackson's
method is just one of MANY variations on how to achieve polymerization
of polymethymethacrylate.

Diane controls her polymerization after embedding, by letting embeddded
bone sit overnight BEFORE you polymerize in a 37C waterbath.  You can
pull a vacuum for a few hours before letting it sit at room temperature
not under vacuum, in a tightly sealed embedding container.
This seems to stabilize the plastic, probably allows some not so apparent
polymerization to occur, and prevent bubbles from forming when you go
to waterbath for final polymerization.  With really large bone specimens,
you may need to layer embed, add some embedding mixture, let it polymerize,
add another layer, and repeat, etc, etc in order to prevent a mass of bubble
when a large volume of embedding mixture polymerizes all at once.  Been
there, had it happen, cried a lot!  I don't know if Peter's method is
designed for really large specimens, Diane's can be used for tiny to
very large.

Her method works very well, is pretty much trouble free.  I have never
tried adding nn dimethylaniline to PMMA mixtures, but have washed
monomer, a tedious, odius, time consuming procedure.  It is much easier
to find a protocol that saves time and money by avoiding washing monomer.
I presume Peter Jackson's tissues are relatively small?  Are they soft
tissues?  I am curious about the size of block he uses?  When he gave his
workshop with Neil Hand, they were not working with large tissue pieces.  I
am not familiar with his new workshop information??

Gayle Callis

<< Previous Message | Next Message >>