SPI-Chem™ Araldite® 6005 Kit
Use Instructions
CAUTION:
Thoroughly read all instructions and warnings on container labels
prior to use of this kit.
Introduction:
"Araldite", a member of a family of thermosetting epoxy resins was first
introduced to the electron microscopy field by Glauert and Glauert1
(1958) who found that the resin had excellent properties as an embedding
medium for electron microscopy. The epoxy resins, in general, demonstrate
low shrinkage (1 or 2%) during polymerization, they are stable during
exposure to the electron beam and are insensitive to oxygen and water.
Embedded specimens are therefore relatively free from polymerization damage
and the plastic does not sublime or otherwise degrade in the vacuum of an
electron microscope. The resin, therefore, provides continual support and
preservation of fine specimen detail.
The mechanical properties of the cured resins are functions of the resin
monomer and the curing agents which are components of the kit are described
below:
- "Araldite 6005" resin is a
low molecular weight, low viscosity, unmodified epichlorohydrin-bisphenol
A condensation product.
- Dodecenyl Succinic Anhydride
(DDSA), a cross-linking agent that is the anhydride of substituted
dibasic succinic acid, and is commonly referred to as the "hardener" or
"curing agent".
- N-benzyl, N-N-dimethylamine
(BDMA), a diamine used as a "catalyst" or "accelerator" which acts as
an end-to-end linking agent.
- Dibutyl phthalate (DBP), a
"plasticizer" which imparts an elastic property to the cured resin.
Formulation:
The usual proportions used to form the cured solid resin are as follows:
Resin Component Typical Medium Hardness Formulation
Araldite 6005 10.0 ml
DDSA 10.0 ml
BDMA 0.5 ml
DBP 0 to 3.0 ml
For additional formulations, the original work of
Mollenhauer2 (1959) should be consulted.
Mixing of Resin Components
The preparation and mixing of the resin components must be thorough and
performed only in a properly functioning laboratory fume hood. With time,
some of the components will separate, so additional mixing is necessary just
prior to the time the resin mixture is to be used.
The cutting properties of the final polymer depend on the components of the
resin monomer mixture. The use of dibutyl phthalate (DBP) materially aids
the cutting properties of the polymerized resin. Although the amount of the
DBP added to each mixture should be optimized for a given specimen and/or
experimental condition, DBP is generally used in the proportion of about 0.5
to 2.0% of the monomer mixture. Likewise, it is desirable that only the
minimum amount of accelerator (BDMA) be used, and should be added just prior
to actual use of the resin mixture. A plastic mixture to which accelerator
has been added cannot be kept unrefrigerated for more than several hours
before it begins to polymerize.
Sample Preparation:
DEHYDRATION:
Dehydration in a graded series of aqueous ethanol solutions (up to 100%
ethanol), followed by propylene oxide or dehydration in acetone has been
assumed.
Infiltration:
Infiltrate using the complete resin mixture in the following schedule:
(a). 2:1 ratio of solvent (propylene oxide or
acetone) to resin mixture with
accelerator, for 1 hour at room temperature (~25°C).
(b). 1:2 ratio of solvent (propylene oxide or acetone) to resin mixture
with accelerator, for 1 hour at room temperature.
(c). 100% resin mixture with accelerator, for 1-2 hours at room temperature.
For some tissues it may be advantageous to apply a vacuum to the sample in
the 100% resin mixture for about 5-10 minutes or until bubbles from the
tissue are no longer visible. Soaking the tissue for periods longer than
that outlined above, generally does not improve tissue preservation. If
materials science samples are being embedded, such as porous ceramics or
catalyst samples, then the vacuum impregnation is almost certainly going to
be important.
EMBEDDING:
Place sample in flat embedding molds or capsules containing the fresh resin
mixture and orient the sample if necessary. An identification code can also
be added to the embedding medium in the mold or capsule.
CURE:
Gently place the embedments in the oven to cure overnight (8-16 hours)
at 80°C.
References:
1Glauert, A.M., and Glauert, R.H., J. Biophys.
Biochem. Cytol. 4, 191 (1958).
2Mollenhauer, H.H., J. Biophys. Biochem.
Cytol. 6, 431 (1959).
When things go wrong:
We are talking now about a resin that won't cure "properly".
Yes, this does happen and we hope that these further comments,
based on our own years of experience might be helpful to someone
having such problems.
The number one reason for such problems is improper dehydration.
Yes, the entire specimen being embedded must be completely dehydrated and
if there are still trace amounts of moisture remaining, the block will not
sure. If the block tends to cure overall, but is not curing in the
vicinity of the specimen, then that is almost certainly the explanation
for the improper curing. Some specimens are much more difficult to
dehydrate than others, therefore if yours are in that category, you
should consider one of the partially water soluble resins, such as
L. R. White,
Unicryl, or
Monostep. Or if you have a
really difficult to embed system, try the
SPI-Chem™ Low Acid GMA, it is
completely water soluble and no dehydration is needed.
There can be real reasons why individual components are not any
longer "good". In this kit formulation, the DDSA is the most moisture
sensitive, in that moisture will rather quickly be absorbed by the
anhydride, and this will result in a substantial increase in the free acid.
The first signs of this happening are the presence of small "pores" in the
cured resin and in the extreme, the resin won't cure at all. The reaction
is not reversible, and unlike come components, where moisture can be driven
off by the application of some heating, in this case the reaction
is not reversible.
In terms of shelf-life, the BDMA is the "first to go". It releases
ammonia, but in most instances, one can compensate for this by adding
an excess of BDMA to their formulation. The only downside to doing
this is that the block cures to a much darker color.
Another concern of many users is that when the DDSA is added to the
DMP-30, the mixture can result in a "reddish" color, but upon curing,
the reddish color disappears and the block does cure to its normal
color and clarity. This variation in color effect at this point is
due to some trace level impurities present in the DDSA. One can work
hard to remove them, so that this color effect is not experienced, however,
the main effect of such removal is to increase substantially the cost of
the DDSA to the user. No one has ever reported a single time any negative
effect of this temporary color effect. So we make comment about the color
effect only so that if you see it, you don't think this is in of itself
a cause for concern.
And remember that like everything else in life, nothing is "forever" and
that is certainly true of organic chemicals. Be sure to follow recommended
storage instructions to realize maximum rather than minimum shelf life.
When in doubt, it is usually cheaper to dispose of out of date chemicals
rather than to risk erroneous results from an important experiment. For
that reason, we believe it is always better to order amounts that are more
closely tied to short and medium term needs rather than to purchase
quantities that could last a life time.
Disclaimer:
The information given in this bulletin is to the best of our knowledge
accurate, but no warranty is expressed or implied. It is the user's
responsibility to determine the suitability for his own use of the products
described herein; and since conditions of use are beyond our control, we
disclaim all liability with respect to the use of any material supplied by
us. Nothing contained herein shall be construed as a recommendation to
practice any patented invention nor as a recommendation to use any product
or to practice any process in violation of any law or any government
regulation.
Caution! Do not use SPI Supplies products or materials in applications
involving implantation within the body; direct or indirect contact with the
blood pathway; contact with bone, tissue, tissue fluid, or blood; or
prolonged contact with mucous membranes. Products offered by SPI Supplies
are not designed or manufactured for use in implantation in the human body
or in contact with internal body fluids or tissues. SPI Supplies will not
provide to customers making devices for such applications any notice,
certification, or information necessary for such medical device use required
by US FDA (Food and Drug Administration) regulation or any other statute.
SPI Supplies and Structure Probe, Inc. make no representation, promise,
express warranty or implied warranty concerning the suitability of these
materials for use in implantation in the human body or in contact with
internal body tissues of fluids.
The information and recommendations set forth above are taken from sources
believed to be accurate as of the date hereof, however SPI Supplies and
Structure Probe, Inc. make no warranty with respect to the accuracy of the
information or the suitability of the recommendations, and assume no
liability to any user thereof. The information contained in this sheet does
not constitute a hazard assessment and should not be used in place of the
user's own assessment of work place risks as required by other health and
safety legislation.
Be aware of the Structure Probe, Inc. Copyright Policy.
Structure Probe, Inc. grants a nonexclusive license to make unlimited copies
of this instructions-for-use sheet for internal use only. Quite obviously,
this information would pertain only to this material when purchased from SPI
Supplies as product from other sources, with other ingredients and impurity
levels could have substantially different properties.
SPI-Chem™ is a trade name of Structure Probe, Inc. and SPI Supplies.
Araldite® is a product of the Ciba Products Corporation.
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Monday December 01, 2008
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