231
Experiments on embedding media for electron microscopy
By S. M. MCGEE-RUSSELL and W. C. DE BRUIJN
(From the Electron Microscopy Laboratory, Virus Research Unit, Medical Research
Council Laboratories, Carshalton, Surrey, England, and Centraal Pathologisch
Laboratorium, Ziekenhuis Dijkzigt, Rotterdam, Holland)
With 2 plates (figs. 2 and 3)
Summary
Experiments were conducted to determine the feasibility of improving the trimming
and sectioning properties of the polymers glycol methacrylate (Rosenberg, Bartl
and LeSko, 1960) and durcupan (X133/1097) (Staubli, i960) by combining them with
other resins, in particular epon 812. We have also attempted to determine the value
of comparative studies of tissue given different fixation and dehydration treatments,
prior to embedding in the same final embedding medium.
Introduction
OVER the last 5 years new embedding materials have been developed for
electron microscopy. Certain of these media employ as an important element
in the final embedding mixture a water miscible component, which has been
used by some workers to replace the conventional alcohol dehydration step.
Progress in this field has recently been reviewed by Glauert.3 Of the characteristics of the water miscible media, some offer possible improvement over
methacrylate, but other features leave much to be desired when compared
in ease of handling with epon or araldite. For example: glycol methacrylate
(GMA) makes an extremely hard and brittle final block and the polymer
swells considerably on contact with water; with durcupan it is not easy to
obtain a block which is of satisfactory composition and hardness for cutting.
Bernhard and Leduc1 have achieved some success by adding a proportion
of the usual methacrylate mixture to glycol methacrylate. We collaborated
in 1961 and 1962 in experiments designed to test the value of the newer
embedding media in our hands, and we have attempted to improve the cutting
properties and preservation of the tissue in the final block in 2 principal ways:
(a) by using the water miscible resin component as a dehydrating agent,
and embedding the tissue in another monomer mixture of satisfactory
properties with which it is miscible;
(b) by making mixtures of the water miscible components with other water
immiscible or partly water miscible plastics, and determining the polymerization characteristics and other properties of the mixtures.
The results reported in this paper are largely concerned with the data
obtained under the heading (b).
During these investigations it was necessary to obtain some basic information regarding the miscibility with one another of the reagents often used in
[Quart. J. micr. Sci., Vol. 105, pt. 2, pp. 231-44, 1964.)
232
McGee-Russell and de Bruijn
embedding technique for electron microscopy. From these studies it has
become clear that there, are interesting means available for the truly comparative study of the effects of the dehydrating and embedding steps upon the
final appearance of the tissue in the electron microscope.
Results
Miscibility tests
The tests were performed by placing 3 ml of each of 2 components in a
test-tube, shaking gently until the contents were mixed, and standing at room
temperature for 24 h. Each test-tube was inspected regularly for any signs
of layering or inhomogeneity. Of the components tabulated here (table 1),
most were well miscible, and showed no signs of layering after 24 h.
TABLE I
Miscibilities
of reagents in common use in embedding for electron
133/
m
1
8
microscopy
0
2
>
Epon 812
Epon ' C
X133/2097
++
++
GMA
Propylene Oxide
Methanol
' Methacrylate1
Well miscible = ++
Immiscible (milky mixture which layers) = Miscible (after vigorous shaking, with no subsequent layering) = +
From these observations it would appear likely that one may dehydrate
tissue with either GMA or X133/2097 and embed the tissue in either methacrylate or epon or vestopal. We have tested this possibility using tissue fixed
in either osmium tetroxide or formalin, dehydrated in graded steps of GMA
or X133/2097, and passed via a 1:1 mixture of dehydrating agent and
embedding material, into the final supporting medium. Analysis of the
morphological effects of these treatments is not complete, but thus far no
special difficulties have been encountered in the normal technical procedures,
and tissue preservation seems adequate.
Polymerization mixtures
There is clearly a very large number of possible combinations which could be
tested. Indications of the possible value of different groups of combinations
Embedding media for electron microscopy
233
can however be obtained by studying the results of mixing together the
different complete resin mixtures recommended in the literature. We chose,
arbitrarily, to mix them in the proportion 1:1, and we used the following
basic 'mixes':
1. Epon ' C = EC = Luft's complete resin mixture, 5A plus 5B with
2% v/v DMP 30. (Luft.)4
2. 'Bartl' = BC = 97 parts commercial GMA (Rohm & Haas) plus 3
parts of a 16-66% w/v (NH4)2S2O8 solution (to give 0-5% final concentration). (Rosenberg, Bartl, and Lesko.)7
3. 'Methacrylate' = MC = 9 parts butyl methacrylate plus 1 part methyl
methacrylate (stabilizer removed) plus 2% w/v benzoyl peroxide.
4. 'Leduc' = LC = 7 parts 'Bartl' plus 3 parts 'Methacrylate'.
5. 'Staubli' = SC = 2-5 parts Xi33/2097 plus 5-5 parts hardener 964 plus
o-6 parts accelerator 960. (Staubli.)9
6. 'Vestopal' = VC = 50 parts vestopal W, 0-5 parts initiator, 0-5 parts
activator. (Ryter and Kellenberger.)8
The mixtures were made in accordance with the original published methods
at the concentrations stated above. The resin mixtures listed above were
mixed thoroughly in pairs 1:1 in test-tubes, in accordance with table 2, and
TABLE 2
Code designations of mixtures tested; proportions
EC
EC
\
BC
MC
LC
BC
EB
MC
LC
SC
VC
ES
EV
EL
EM
Of no Of no
value value
Of no
\
t value
BS
BV
MS
MV
^ X
LS
LV
SV
SC
EC='EponC
LC='Leduc'
1:1
BC='Bartl' MC ='Methacrylate'
SC ='Staubli' VC='Vestopal'
For components of basic mixes see text
inspected for any signs of layering after several hours at room temperature.
Each resultant double mixture was given the designation listed in the table.
Most pairs proved to be well miscible, but MV and SV show layering after
24 h at room temperature. ES is a rather viscous combination.
We consider the low temperature (37° C) overnight incubation step, which
appears in many impregnation procedures, a very significant one. It ensures
maximum impregnation of the tissue with plastic of somewhat lowered
viscosity, and also initiates polymerization gently. Omission of such a step
234
McGee-Russell and de Bruijn
of 'prolonged soaking' at 37° C or similar temperature has a direct effect, as
one would expect, upon the final curing time, by whatever means. We therefore routinely included in our test procedures a stage at 37° C. Capsules
were filled with the double mixtures, incubated overnight at 370 C after
capping, and then finally polymerized either in an incubator at 6o° C, or
under UV at 4 0 C. The results are given in tables 3 to 5 (appendix). When
these experiments are carried out in glass test-tubes in larger quantity, rather
than in gelatin capsules, results are similar, but not identical. This is most
noticeable in the cases where water is added to the resin mass, and it seems
likely that the gelatin capsule acts as a significant 'sink' for the water present
during polymerization. The tabulated results are based on the results obtained
in capsules, as capsules are part of the normal working procedure in electron
microscopy. There are often differences between rates of polymerization
under UV or at 60° C. The precise character of the blocks during trimming
and sectioning can be slightly different after the two treatments. However,
UV and 60° C treatments are not invariably different in timing or results (e.g.
LC table 3).
Conditions for UV polymerization
De Bruijn (unpublished) had previously demonstrated that an apparatus
of the type illustrated in fig. 1 could be used at 4 0 C and below to achieve
good polymerization of methacrylate in gelatin capsules. The apparatus is
based on that published by Miiller.6 At — io° C the rise in temperature
within the polymerizing plastic in the capsules was held below 170 C, and it
is likely that use of a cold room at — 40° C would maintain the temperature
of tissue in the capsule at o° C or below. This would be valuable for histochemical studies of enzymatic activity. As the possibility of enzyme histochemistry was one of our reasons for investigating the water miscible resins,
we felt that it was necessary to include UV polymerization in our studies.
Either a Philips HPK 125 type 126036 ultraviolet lamp or a Philips ultraviolet MLU 300 W 220-240 V type 57265 F/28 may be used in this type of
apparatus. In all the experiments reported in this paper we used the latter
type of lamp. The fan may be placed either above the lamp or below the
capsules. The preferred direction for the air flow is towards the lamp, away
from the capsules. Lamp to capsule distance was minimally 10 cm.
In table 3 we give the data for curing times and condition of polymer
mixtures in order to offer a guide to other workers, but no more than a guide.
Clearly the exact timings will vary from laboratory to laboratory, in accordance
with uncontrolled variables. Curing times of over 10 h with UV and of over
48 h at 6o° C we generally consider as unsuitable for routine procedures.
Polymer mixtures which were opaque, cloudy, or inhomogeneous were not
regarded as suitable for further study, although it is not proven that cloudiness
of supporting medium will affect the preservation of tissue or the electron
microscopic image of such tissue. Nor can it be assumed that a cloudy polymer
is unsuitable for sectioning, as shown by the mixture EV (table 6).
Embedding media for electron microscopy
235
FIG. I . Diagram of apparatus suitable for UV polymerization of methacrylate, glycol
methacrylate, durcupan, epon, or vestopal blocks, or mixtures of resins.
A—lid with louvres for free ventilation, shiny metal.
B—cylinder of shiny metal alloy for maximum reflection.
c—capsule containing polymer.
D—Philips HPK 125 type 126036 UV lamp, or a Philips MLU 300 W 220-240 V type
57065F/28 UV lamp.
E—cork ring with depressions to take capsules (thick perspex tends to distort under prolonged UV).
F—small fan (diameter c. 20 cm). With small fan the diameter of the apparatus can be held
to 21 cm. with the smaller HPK 125 lamp.
G—ventilation slots in base of cylinder for ingress of air.
1—retort stand with adjustable bossheads which permit immediate adjustment of relative
distances of elements of apparatus.
J—Philips choke for UV lamp HPK 125. Choke type HP(L) 125 W 58213 AH/00 220 V
50 cycles 1-15 amps. (With the other larger lamp no separate choke is required.)
s—slot in the side of the metal cylinder for entry of supports.
Comments on experimental mixes in table 3
The most promising double mixture observed in the experiments tabulated
is the mixture 1:1 epon C and Leduc (EL), which appears to have very much
236
McGee-Russell and de Bruijn
the consistency of epon, but also contains the water miscible GMA. Since
epon 812 itself contains a high proportion of water miscible resin (30% aquon,
Gibbons2), it seemed likely that a satisfactory polymer with some water content might be made of this combination. We have tested mixtures with final
water contents of between o to 20%. The miscibility of the components
remains unaffected up to about 10% added water. Above this concentration,
separation of the components of the mixture becomes noticeable. The mixtures are designated EL95, EL90, and EL 80 , for final water contents of about
5%> Io%> a n d 20%, and the polymerization data are listed in table 3. One
may be able, by the use of a mixture like EL, to support tissues in plastic for
thin sectioning with an appreciable quantity of water (optimally 5 to 10%)
still present.
A second important feature is that the mutual miscibility of the GMA,
methacrylate, and epon components and the satisfactory nature of the final
polymer EL, permits one to formulate experiments which will demonstrate
comparatively the effect on the final image of different stages and reagents
used in processing. Material can be fixed in the same manner, divided into
portions, and submitted to different dehydration procedures, prior to embedding in the same final supporting medium. We have conducted experiments
along these lines, which suggest that useful information can be obtained from
such studies.
One would not necessarily expect a complex mixture like EL containing
3 different plastics to form a better polymer than a mixture of like plastics,
for example of epoxy-type resins. It is interesting, therefore, to note the
results obtained for the combination ES containing epon 812 and the water
miscible epoxy resin X133/2097. The components are well miscible, but
form a viscous combination, which is difficult to handle. However, better
penetration into the tissue block may be assured by an antemedium of
1,2 epoxy-propane, with which it is well miscible. Reduction of the hardener
in either the 'Staubli' or the 'epon C might also reduce viscosity. The consistency, trimming and cutting properties of the final polymer are much more
encouraging than for the original Staubli mixture, and more closely approach
epon. Very recently, W. Staubli10 published a procedure in which X133/2097
(durcupan) is used as a dehydrating agent prior to embedding in araldite.
We did not test combinations of X133/2097 with araldite, as we assumed that
epon would offer us the same features as araldite.
Following the reasoning applied to the mixture EL, we have tested whether
it is possible to polymerize ES mixtures with a moderate water content of
between o to 20%. The mixtures for which results are listed in table 3 are
designated ES 95 , ES90, ES80, for final water contents of about 5%, 10%, and
20%. We also tested the effect of added water upon the original mixture of
Staubli9 by diluting the component X133/2097 (durcupan A) with appropriate quantities of water. The presence of water in the final original mixture
of Staubli does not prevent polymerization, but generally gives a polymer
which is too soft for sectioning. From the data in table 3 it is clear that above
Embedding media for electron microscopy
237
a final concentration of about 10%, water also makes the mixture ES too
soft. But below 10%, especially if only 5% or less is present, ES is a useful
polymer which trims and sections easily. There are some indications that
water is actively excluded from resin masses,..particularly.during heat polymerization, and subsequently lost by evaporation. One cannot therefore
assume that the entire amount of added water is present in the final cured
mass of resin. The softness of the ES mixtures with a higher water content
may perhaps be overcome by using a harder epon C mixture, or by using
a ratio E: S of 2:1 instead of 1: i, but we have not yet tested this.
All our observations emphasize, a point which appears throughout the
experiments: complex mixtures of plastics, hardeners, plasticizers, and catalysts do not behave in a predictable manner, and their properties can only
be evaluated by empirical test. For example, although, as shown in tables
1 and 3, methacrylate and vestopal are immiscible, the mixture _LV, which
contains glycol methacrylate as well as methacrylate and vestopal, does not
show the layering and inhomogeneity which might be expected, either on
mixing or on polymerization (table 3), and sections well (table 6).
It seems likely that the polymerization of one catalysed monomer may
directly affect the polymerization of another associated with it. Thus, as
shown in table 4, where we assess the percentage of polymerization after
a constant time (15 h) at 37° C, there may be mutual acceleration in the
combinations of methacrylate with GMA (LC), and of both with epon (EL).
Our assessments are based on the proportion of the resin mass in the capsule
which became firm, usually in the lower part of the capsule, the rest remaining
liquid. Furthermore, from this table it can be seen that the other epoxy resin,
X133/2097, in combination with either methacrylate (MS) or glycol methacrylate (BS), and catalysts, suffers inhibition, but that this is much reduced
when it is mixed with both together (LS). It is possible that there may be
direct interaction between polymerizing chains. It is also possible that catalysts may interact directly with each other. It might be expected that catalysts for one plastic could cause polymerization in another. In order to go
some way to disentangle these interactions, we obtained the data listed in
table 5. From this table and other experiments we can draw the following
conclusions:
(a) Neither benzoyl peroxide nor ammonia persulphate is an effective
catalyst for epon 812 alone.
(b) In increased concentration, neither benzoyl peroxide nor ammonia
persulphate promotes the polymerization of a 1:1 mixture of GMA and
epon 812 since the times given in table 5 are much the same as those
given in table 3 for the control mixes.
(c) There is no direct interaction between GMA and epon 812 during
polymerization.
(d) DMP 30 is not an effective catalyst for X133/2097 or for mixtures of
X133/2097 and epon 812. The addition of X133/2097 alone to epon C
therefore tends to slow down its normal rate of polymerization.
238
McGee-Russell and de Bruijn
(e) Accelerator 960, the normal catalyst for X133/2097, is also a catalyst
for epon 812, particularly when in combination with its curing agents
DDSA (dodecenyl succinic anhydride) or MNA (methyl nadic anhydride). Hence it also catalyses mixtures of epon and Xi33/2097.
(/) DMP 30 is not an effective catalyst for vestopal W nor is accelerator
960. It can be shown that vestopal W alone can be hardened completely
by 15 h at 370 C followed by 4 h under UV, and the times for the normal control mix VC listed in table 3, are shorter than those in table 5
with added DMP 30.
(g) Benzoyl peroxide is the normal initiator for the usual vestopal mixture,
but it is interesting to note that a doubled concentration (2%) of ben2oyl
peroxide alone produces a hard polymer in less time than the normal
proportions of initiator and activator.
(h) Cobalt naphthenate, the activator of vestopal W, is not an effective
catalyst for the Staubli mixture.
(i) As a mixture of X133/2097 alone and 'Vestopal' hardens quickly without layering, but the SV mixture layers considerably during curing, it
may be inferred that the hardener DDSA is immiscible with vestopal.
This is confirmed by direct experiment.
Other similar inferences may be drawn from the tables, and tested. The value
of the tables lies in the multiplicity of treatments which are shown to be
available to prepare tissue for electron microscopy. Such a variety of techniques may be of considerable help in studying refractory tissues and should
also be valuable in forming assessments of the results obtained by current
procedures.
Trimming and sectioning properties
Mere polymerization of a mixture does not constitute an adequate indication of its suitability for histological electron microscopy. It is necessary to
embed well-known tissues in the media, cut sections, and assess the quality
of the supporting medium by the study of micrographs. This, however, is an
extremely time-consuming task, if applied to many different mixtures. It is
possible to shorten the task by considering some ancillary features which are
more quickly assessed. Certain of these may be considered under the heading
'trimming and sectioning properties'. The criteria are highly subjective, but
are descriptive of properties rapidly appreciated by anyone used to handling
blocks for ultra-microtomy. Table 6 lists our appreciation of the experimental mixtures in these terms.
FIG. 2 (plate). Typical complex cytoplasm of Krebs 2 ascites tumour cell from sample
embedded in epon. (Fixation: veronal acetate buffered osmium tetroxide with added calcium
chloride (pH 73). Dehydration: alcohol series. Embedding medium: epon. Section stained:
uranyl acetate.) Note the preservation and characteristic distribution of smooth and rough
membranes (ser and rer); virus-like particles (yip); mitochondria (m); and other elements.
FIG.
2
S. M. M c G E E - R U S S E L L and W. C. DE BRUIJN
Fie. 3
S. M. McGEE-RUSSELL and W. C. DE BRUIJN
Embedding media for electron microscopy
239
All assessments of trimming properties were made using fresh razor blades
on cold blocks, i.e. blocks which had been at room temperature for many
hours. All assessments of sectioning quality were made by the same observer
working with a Huxley ultramicrotome and glass knives, using 20% acetone/
water in the boat. It is worth pointing out that the elaborate saws and grinding
tools which have been advocated by some workers are not necessary to trim
even the hardest blocks. If a refractory block is returned to the 6o° C oven
until warm, or washed in warm running tap water, and trimmed shortly after,
all trimming and shaping operations can be done with a single fresh razor
blade. Sectioning should be delayed after such a warm trimming procedure
until the block has thoroughly cooled.
Effects on embedded tissues
We have not completed a full survey of osmium, formalin, and permanganate fixed tissues in the experimental mixed embedding media. However,
as mentioned above, and as can be seen from the tables, the most promising
mixture of all is that designated EL, epon/Leduc, containing Luft's4 complete epon mixture and Bernhard and Leduc's1 mixture of conventional
methacrylates and glycol methacrylate. By examining Krebs 2 ascites tumour
cells embedded in EL as test objects, after fixation by centrifugation through
each of the 3 fixatives, we are satisfied that this medium does not produce
polymerization damage. Membrane preservation is as satisfactory as it is
after using epon itself. The medium 'clears' in the beam to about the same
extent as epon, and may be used satisfactorily on naked grids, for it is of high
stability in the beam. It is less viscous than epon CC and therefore easier to
handle during infiltration and embedding. Experiments on different methods
of pre-treatment before final embedding in EL show that with conventional
osmium fixation and dehydration prior to embedding in EL, there is virtually
no difference from the images seen in ordinary epon, figs. 2 and 3. Preservation as good as any reported in the literature, is obtained after formalin or
permanganate. Given a final supporting medium of high stability, which EL
would appear to be, our experiments seem to indicate that the most significant steps after fixation are those involved in dehydration. There is a suggestion that the embedding medium may affect, to some extent, the final
'staining' or 'contrasting' of the embedded tissue with uranyl acetate, as
compared with tissue in epon, but this would seem to apply mainly to large
lipid containing droplets. The majority of cellular elements are stained
Fie. 3 (plate). Typical complex cytoplasm of Krebs 2 ascites tumour cell from sample
embedded in mixture EL (see text). (Fixation: veronal acetate buffered osmium tetroxide
with added calcium chloride (pH 7'3). Dehydration: alcohol series. Embedding medium:
EL (1:1 mixture of Epon and 'Leduc'). Section stained: uranyl acetate.) Compare the
preservation of the typical elements: ribosomes (r); smooth membranes (ser); rough membranes (rer); virus-like particles (vlp); and mitochondria (m); with the same objects in fig. 2
(plate). A small part of the nucleus is also visible, showing the same type of preservation seen
in epon.
34°
McGee-Russell and de Bruijn
normally. Tissue embedded in EL, like epon, can be stained satisfactorily
with either Nile blue or Sudan black solutions for light microscopy (McGeeRussell and Smale).5
Discussion
We have demonstrated that epon can be mixed with certain other plastics
during dehydration sequences or in the final embedding medium. Tissues
may therefore be brought through water miscible plastics into a final medium
of epon, or water miscible plastic mixed with epon, so improving the sectioning qualities of otherwise difficult media. In comparing dehydration sequences
experimentally, it is sometimes more satisfactory if the final embedding
medium is exactly the same. The mixture designated EL in this paper is
suitable for experiments of this kind. We have shown that some combinations
of plastic will tolerate the presence of water in the final embedding medium.
This may mean that a wide range of histochemical techniques can be applied
fruitfully to sections of tissue embedded, after suitable processing, in epon, or
in combinations of epon with other plastics.
We wish to thank Mr. C. J. G. Smale and Miss H. Belham for their valuable
technical assistance. We are grateful to Dr. F. K. Sanders for providing many
samples of Krebs 2 ascites cells, and for the hospitality he extended to W. C.
de Bruijn within his Unit, which enabled us to continue a stimulating collaboration; also to the Medical Research Council for the facilities of their
Laboratories at Carshalton. W. C. de Bruijn's visits to this country in 1961
and 1962 were made possible through the generosity of the Dutch Organisation for Pure Scientific Research, Z.W.O., 's-Gravenhage, Holland, and the
City Council of Rotterdam.
References
1.
3.
3.
4.
5.
6.
7.
8.
9.
10.
Bernhard, W., and Leduc, E. H., 1962. Personal communication.
Gibbons, I. R., 1956. Nature, Lond. 184, 375.
Glauert, A. M., 1962. J.R. micr. Soc. 80, 269.
Luft, J. H., 1961. J. biophys. biochem. Cyt. 9, 409.
McGee-Russell, S. M., and Smale, N. B., 1963. Quart. J. micr. Sci. 104, 109.
Miiller, H. R., 1957. J. Ult. Res. 1, 109.
Rosenberg, M., Bartl, P., and Lesko, J., i960. Ibid. 4, 298.
Ryter, A., and Kellenberger, E., 1958. Ibid. 2, 200.
Staubli, W., i960. C.R. Acad. Sci. Paris 250, 1137.
1963. J. Cell Biol. 16, 197.
Embedding media for electron microscopy
241
Appendix
TABLE 3
Polymerization properties of mixtures
Mixture
Control mixes
EC
BC
Condition after
IS h at 37° C
Subsequent
polymerization
time under UV
slightly syrupy
bottom half hard,
top syrupy
hard in 2 to 4 h
hard in 2 to 4 h
Subsequent
polymerization
time at 60° C
hard in 36 h
hard in 6 h
The polymerization times of this mix depend on the source of the GMA. Commercial
GMA appears often to have inhibitors present. The times given here apply to a sample
of uninhibited GMA very kindly given to us by Dr. D. Inman, who made it himself.
hard in 2 to 4 h hard in 4 h even
MC without
hard
even without
without stage at
hydroquinone
stage at 37° C
37° C
liquid
MC with
requires over
requires over 72 h
hydroquinone
12 h
bottom hard, small
LC
hard in 2 to 4 h hard in 2 to 4 h
liquid top layer
The polymerization times of this mix vary considerably according to the source of
the GMA and the presence or absence of inhibitor in the methacrylate.
fairly hard
hard in 2 h
hard in 2 h
SC
irregular polymeriza- hard in 4 to 6 h hard in 6 h
VC
tion varying from
25 to 75%; top
layer syrupy
Experimental mixes
syrupy
hard in 2 to 4 h 90% hard in 24 to
EB
36 h; 10% top layer
still liquid after 72 h
hard in 2 to 4 h hard in 18 h but
bottom half hard
EM
and milky, top
but milky
milky
half syrupy
fairly hard
hard in 2 h
hard in 10 to 12 h
EL
A top layer develops on this mix after shorter polymerization times, which appears
to be a slow fraction present in the GMA (cf. BC + LC). The depth of the layer
follows the proportion of 'Leduc' (LC) in the mix. This does not appear to be due
to immiscibility, but to varying rates of polymerization of individual components.
hard in 2 to 4 h hard in 2 to 4 h
gelatinous
ES
liquid
BS
hard in 4 to 6 h requires over 48 h
liquid
MS
hard in 2 to 4 h gelatinous and milky
after 48 h
but milky
bottom 75 % hard,
LS
hard in 2 to 4 h bottom fairly hard,
top syrupy
syrupy top after 48
h; after 72 h hard,
excluding top layer
of Staubli-like
material still liquid
EV
requires over 8 h hard in 24 h
slightly milky blue
to polymerize
bottom layer with
completely.
syrupy (epon) top
8 to 12 h
layer of about 20%
McGee-Russell and de Bruijn
242
T A B L E 3 (cont.)
Condition after
15 h at 37° C
liquid
layered; bottom
layer milky blue
and viscous to hard
liquid
layered, milky gelled
bottom layer of
vestopal. Syrupy
top (Staubli)
bottom half gelled,
top syrupy
bottom half gelled
and slightly cloudy,
top syrupy
marked layering,
still liquid
Mixture
BV
MV
LV
SV
EL 95
EL 90
EL 80
ES 95
ES 90
ES 80
Subsequent
polymerization
time under UV
hard in 4 to 6 h
not observed
Subsequent
polymerisation
time at 6o° C
hard in 24 to 36 h
not observed
hard in 6 to 8 h
not observed
hard in 24 h
not observed
hard in 2 to 4 h
hard in 24 to 36 h
hard in 2 to 4 h
hard in 24 to 36 h
hard in 4 h
hard in 48 h with
tendency to exclude
excess water
hard in 32 h
hard in 32 h
hard* in 32 h
hard in 6 to 8 h
hard in 8 h
hard* in 8 h
gelatinous
gelatinous
gelatinous
hard* = completely polymerized but insufficiently hard for sectioning.
TABLE 4
The effects of mixing media on polymerization
7
IO
7
—
IO
3
7
—
LC
EM
EB
EL
ES
EV
BS
MS
LS
SV
MV
BV
LV
2 1 1 1
IO
—
IO
IO
IO
IO
3
3 1 1 11
3
22222
IO
MC
BC
EC
SC
VC
3 3 33
—
VC
Code
designation
1 3 1 11
7
IO
1 3 1 11
3
IO
1
1 1 1 12
IO
1 1 1 31
MC
Parts of:
BC
EC
SC
Estimated % (v/v) degree of
polymerisation observed after
overnight at 37° C
100%
50%
0%
100%
25-75%
90% (expectation: 65%)
50% (expectation: 50%)
o%(
„
25%)
95% (
„
45%)
0% (
„
50%)
80% (
„
30%)
0% (expectation: 70%)
0% (
„
100%)
75% (
„
95%)
— not miscible
— not miscible
0% (expectation: c. 50%)
0% (
„
c. 70%)
Embedding media for electron microscopy
243
TABLE 5
Polymerization properties of components
Combinations
Epon 812 + 2% w/v Benzoyl peroxide
Epon 812 + 2% w/v (NH 4 ) 2 S 2 O 8
Epon 812 1 part: GMA 1 part +
2% w/v benzoyl peroxide
Epon 812 1 part: GMA 1 part+
2% w/v (NH 4 ) 2 S 2 O 8
Epon 812 1 part: X133/2097 1 part
+ 2% v/v DMP 30
Epon 'A' 1 part: Xi33/2097 1 part
+ 2% v/v DMP 30
Epon 'B' 1 part: X133/2097 1 part
+ 2% v/v DMP 30
Epon 'C 1 part: X133/2097 1 part
+ 2% v/v DMP 30
Epon 812 1 part: X133/2097 1 part
+ 2 % v/v accelerator 960
Epon 'A' 1 part: X133/2097 1 part
+ 2 % v/v accelerator 960
Epon 'B' 1 part: X133/2097 1 part
+ 2% v/v accelerator 960
Epon ' C 1 part: X133/1097 1 part
+ 2% v/v accelerator 960
Epon 812 + 2% v/v accelerator 960
Epon 'A' + 2% v/v accelerator 960
Epon 'B' + 2% v/v accelerator 960
Epon 'C' + 2% v/v accelerator 960
Vestopal (Weich)+2% v/v DMP
30
Vestopal (W) + 2% v/v accelerator
960
Vestopal (W) + 2% w/v benzoyl
peroxide
Staubli + 2% v/v activator of vestopal cobalt naphthenate
X133/2097: 'Vestopal' = 1 : 1
Condition
after 15 h
at 37° C
liquid
liquid
liquid
Subsequent
polymerization
time under UV
>
10 h
>
10 h
Subsequent
polymerization
time at 60° C
> 8z h
>82h
hard in 6 h
liquid
hard in 2 h but
milky
hard in 2 h but
milky
> 10 h
liquid
> 10 h
>82h
liquid
> 10 h
>82h
liquid
>
> 82 h
liquid
10 h
hard in 6 h
>82h
liquid
> 8h
> 72 h
syrupy
> 8h
> 72 h
syrupy
hard* in 6 to 8 h
> 72 h
syrupy
hard* in 6 to 8 h
> 72 h
liquid
gelatinous
gelatinous
gelatinous
syrupy
>8h
> 72 h
hard* in 6 to 8 h hard* in 48 h
hard in 6 to 8 h hard in 48 h
hard in 6 to 8 h hard in 48 h
hardish in 8 h
> 72 h
syrupy
90% hard
liquid
hard*
>8h
> 72 h
hard in 2 h
hard in 6 h
>8h
hard* in 2 h
> 72 h
hard* in 2 h
hard* = completely polymerized but insufficiently hard for sectioning.
McGee-Russell and de Bruijn—Electron microscopy
244
TABLE 6
Assessment of trimming and sectioning properties of mixtures
Mixture
code
designation
LC
EB
EM
EL
ELe°
ES
ES 90
BS
MS
LS
EV
BV
MV
LV
sv
Trimming and sectioning properties
Hard and brittle. Difficult to trim, with a marked tendency to conchoidal fracturing. Sectionable but not entirely satisfactory.
Rubbery and somewhat soft. Trims easily. Sections excellently.
Hard and brittle. Difficult to trim. Sectionable but inhomogeneous.
Hard; during trimming slightly more brittle than normal epon
(5A:sB). Sections excellently.
Hard; more brittle than normal epon (sA^B), with tendency to
conchoidal fracturing. Sections satisfactorily.
Rubbery and somewhat soft. Trims easily and sections satisfactorily
with some tendency for sections to build up on the knife edge.
Rubbery; less brittle than normal epon (sA:sB). Trims easily.
Sections satisfactorily.
Hard; trims like epon. Sectionable.
Inhomogeneous, not suitable.
Hard. Trims well with some tendency to conchoidal fracturing.
Sections satisfactorily.
Hard. Slightly milky bluish polymer. Trims well. Sections satisfactorily, although inhomogeneous.
Hard and brittle. Difficult to trim, with marked tendency to conchoidal
fracturing. Sectionable, but block hydrophilic, giving 'meniscus
jump'; very careful adjustment of level of meniscus in boat required
for sectioning.
Inhomogeneous, not suitable.
Hard. Trims smoothly and well. Sections excellently.
Inhomogeneous, not suitable (see text).