Anat Embryol (1987) 175:41~421
and Embryology
Review article
The current potential of plastination*
Gunther yon Hagens, Klaus Tiedemann, and Wilhelm Kriz
Anatomisches tnstitut I, Universit/it Heidelberg, Heidelberg, Federal Republic of Germany
Summary. This review surveys the potential of plastination,
a technique of tissue preservation introduced eight years
ago. In this process, water and lipids in biological tissues
are replaced by curable polymer which are subsequently
hardened, resulting in dry, odorless and durable specimens.
The procedure consists of the following steps - fixation,
dehydration, forced impregnation in a vacuum, and harden-
ing. The properties of the finished specimen are determined
by the class of polymer used.
Silicone yields flexible, resilient specimens, allows the
broadest range of application, and provides satisfactory re-
suits with minimum equipment. Specimens plastinated with
an epoxy-silicone copolymer are rigid enough to be pol-
ished, but are not unbreakable. This resin is used for thick,
opaque body slices and showcase specimens. Epoxy resins
are used for thin (2.5 ram), transparent body or organ slices.
They are cast between polyester foils or glass plates and
can be used for histological investigations. Polyester resin
is used for the production of opaque brain slices, which
gives excellent differentiation between grey and white mat-
ter. The application of plastination in research and the pro-
duction of teaching specimens is discussed with special re-
gard to the equipment required, cost, and feasibility of the
processing.
Key words: Plastination - Preservation - Macroscopy -
Technique
Introduction
Anatomists have long sought a technique for the preserva-
tion of soft tissue, producing durable, dry and handable
specimens. Both, Deegener and Berndt (1914) and Hoch-
stetter and Schmeidel (]924) came close to this goal with
their paraffinization technique. Their patent specification
extols the dry texture, natural appearance, and great resis-
tance to mechanical influences of paraffinized specimens.
However, the claim of an unlimited durability was exagger-
OffprilTt requests to: G. v. Hagens, Anatomisches Institut I, Im
Neuenheimer Feld 307, D-6900 Heidelberg 1, Federal Republic of
Germany
* The plastination process is covered by several patents (v. Hagens
1977 ff.), although the production of specimens on a non-com-
mercial basis is virtually unrestricted
Anatomy
9 Springer-Verlag 1987
ated, as paraffinized specimens turned out to be delicate,
heat-sensitive and flammable.
The improved properties of plastinated specimens are
mainly accounted for by the superior qualities of curable
polymers. In the plastination technique, tissue water and
lipids are replaced by cured polymers. The class of polymer
used determines the mechanical (flexible or firm) and opti-
cal (opaque or .transparent) properties of the specimen.
Plastinated specimens are dry, odorless, and durable; they
even retain structural details down to the histological level.
Today, eight years after its introduction (v. Hagens
1977, 1979a, b), plastination is being applied in more than
150 departments of anatomy, pathology, forensic science
and biology all over the world. During this time, diverse
new techniques have been developed, and a vast array of
experience has been accumulated.
This review is intended to give a survey of the applica-
tions of the plastination technique and to serve as an intro-
duction to the technique itself. There are two major areas
of application. For teaching and exposition purposes, dura-
ble specimens of a previously unknown variety and quality
are provided. In research the technique of sheet plastination
allows the arrangement of all tissue components to be stud-
ied in their undisturbed context. This is of major interest
in the borderline area between gross anatomy and histology
with respect to muscular and connective tissue patterns.
The description of the technique given here should illustrate
the actual processing in the laboratory sufficiently well to
answer any question as to its feasibility. However, this re-
view is not intended to serve as a laboratory guide. A step
by step description of the techniques is available in a com-
prehensive technical manual (v. Hagens t985/86).
Applications
Basically, there are four variations of the plastination tech-
nique providing four different kinds of specimens. Silicone
impregnated specimens (Figs. 1-5) are resilient and flexible
and are mainly used in teaching. Specimens produced with
polymerizing emulsions (Figs. 6-8) are as opaque as the sili-
cone specimens but are rigid and to some extent breakable.
The use of this technique is in thick body slices exhibiting
a superb contrast between fat tissue, which show up white,
and all other more intensively stained parenchymas. Trans-
parent body or organ slices (Figs. 9, 10, 13, 14) are produced
with epoxy resins. For research purposes these slices allow
study of the topography of all body structures in an uncol-
412

Fig.l. Fetus, 7th month. Plastinated with silicone S 10

Fig. 2. Uterus from 13th week of pregnancy. Ovary and fallopian tube on top. Plastinated with silicone S 10. The specimen was stained
after previous bleaching
 413

lapsed and non-dislocated state. In addition the specimens
are useful in advanced traning programs in sectional topog-
raphy (resident training in CT and NMR). Opaque brain
slices (Figs. 11, 12) are impregnated with polyester resin;
they allow a unique discrimination between fiber and nucle-
ar areas.
Teaching greatly benefits from all kinds of plastinated
specimens. Epoxy, silicone and polyester-impregnated spec-
imens have received a widespread use and popularity in
anatomy, pathology, zoology and forensic medicine. Such
specimens facilitate teaching considerably. Replacing speci-
mens stored in formalin or alcohol, the dissected material
can literally be grasped without wetting the student's fingers
or their textbooks.
The anatomical department in Vienna was the first user
outside Heidelberg to enrich its gross dissection lab with
skillfully dissected plastinated joints and with body slices
such as shown in Figs. 6 8 (Lischka et al. 1981). In Texas,
the Health Science Center equipped pathology courses with
plastinated specimens because of their ease of handling and
educational value (Bickley et al. 1981; Bickley 1984). An
International Society for Plastination has been formed
(Bickley 1986). Its main functions are to compile and make
available a list of laboratories involved in plastination, to
serve as a forum for the exchange of information about
plastination, to issue a publication, the Journal of the Inter-
national Society for Plastination periodically and to orga-
nize and conduct meetings and workshops.
Plastinated specimens are easily transported and can be
displayed in courses where human organs could not other-
wise be presented in the class room. Our anatomical depart-
ment in Heidelberg uses them in histology classes which
precede the gross dissection lab. They are ideal for examina-
tions, especially when the student has access to a collection
of plastinated specimens.
At present, we supplement our course of topographical
anatomy with plastinated specimens of structures that are
rarely seen in our dissecting room (e.g., deep ligaments;
dilated hearts shown in Fig. 3, color injected kidneys and
placentas, male and female pelvic organs with bladder and
rectum). These specimens are appreciated by the student
as are thick body slices plastinated with PEM (Fig. 8) or
silicone. Transparent body slices (Figs. 9, 10) have been
found to stimulate discussion on topographical questions
among students. It is noteworthy that these slices closely
resemble CT-scan images and are thus of high clinical rele-
vance.
Plastinated brain slices (Figs. 11, 12) are superior to wet
material. Together with stepwise-dissected whole brains,
brain stems, and 1 cm thick slices, all plastinated with sili-
cone rubber, they allow a ' d r y ' neuroanatomy course with-
out formalin-tears. This is an appealing alternative at a
time when the carcinogenic potential of formalin is under
debate (Acheson 1981; Daschner 1983; Pabst 1986).
Most research applications utilize the sheet plastination
technique. Transparent slices of any size can be produced,
limited only by the cutting height of the band saw, with
an optimum thickness of 2.5 mm. In contrast to the trans-
parent loose areolar and adipose tissue (lipid extraction),
the muscle tissues (skeletal, cardiac and smooth), all epithe-
lial parenchymas (of glands, kidney, etc.) and blood stand
out prominently. The course and distribution of connective
tissue strands and fascia can also be easily recognized.
Bones need not be decalcified. The relationships between
connective tissue fibers and bony structures are maintained
(Figs. 10, 14). After filling with a contrasting polymer, the
branching patterns of vessels may be followed down to the
arteriolar level. Moreover, a complete filling of the vascula-
ture expands the tissue to an in vivo-like state. Conse-
quently, the topography of certain structures can be studied
in an uncollapsed and non-dislocated state. Serial slices al-
low the three-dimensional, computerized reconstruction of
complex structures. Plastinated slices represent a valuable
tool for embryological investigations of fetuses which are
too large for paraffin serial sectioning.
Several areas of research can benefit from transparent
specimens. Diagnostic imaging by CT and N M R requires
an ever increasing knowledge of topographical relation-
ships. For instance, magnetic resonance imaging of the orbit
or inner ear using the surface coil brings out topographical
details almost down to the histological level (Schenck 1984;
Schnitzlein and Murtagh 1985). Sheet plastination, giving
excellent detail, becomes available at a time when the quali-
ty of radiological imaging surpasses standard frozen cadav-
er slices. In addition, differences in the topographical rela-
tionships produced by movement (e.g., structures of the
leg in a flexed or extended position) can be investigated.
Another area is the transition between gross anatomy
and histology. As muscle fibers, muscle fiber bundles, col-
lagenous strands, and fascia can easily be identified and
followed within transparent specimens, questions can be
answered concerning muscle and connective tissue fiber pat-
terns and their changes under various conditions. It has
been shown by plastination techniques that the arrange-
ment of uterine muscle fibers is quite dissimilar to what
was generally believed (Br6kelmann and Miiller 1985) and

Fig. 3. Human heart, dilated under pressure, injected with red (arterial) and blue (venous) epoxy resin, impregnation with silicone
S 10. The atrial portions remain flexible

Fig. 4. Dissected arm piastinated with silicone S 10

Fig. 5. A flexible 6 mm-slice of a liver in biliary cirrhosis. Plastination with silicone S 10 using xylene as the solvent. The natural
color preservation results from the use of Kaiserling (1895) fixative

Fig. 6. A reassembled cadaver was sliced every 2 cm and plastinated with a thermosetting epoxy-silicone copolymer (PEM). (Fig. 8
shows a single slice)

Fig. 7. A slice from the cadaver of Fig. 6. In this pre-cured state, the resin mixture obscures the specimen's detail

Fig. 8. The same body slice as seen in Fig. 7, but after curing of the resin mixture. The cured, emulsified resin now combines light
dispersion with transparency as in milky glass. Parenchymal organs can clearly be distinguished from the white adipose tissue
414

can be expected to be different in juvenile, pregnant and
postpartum states. There are several other muscular systems
whose precise fiber pattern is not well characterized, for
example at the uretero-vesical junction and bladder trigone
and neck (Tanagho et al. 1968). Changes in the muscular
syncytium of the heart in systolic state compared to diastol-
ic state (Bargmann and Doerr 1963) await final clarifica-
tion. Textural changes under pathological conditions (e.g.,
heart hypertrophy) and related changes in microangio-
graphic patterns can both be studied in the same specimen.
With respect to connective tissue patterns, many ques-
tions remain unanswered. Within the female pelvis for in-
stance, a variety of connective tissue ligaments, lucia and
septa are generally thought to be responsible for holding
the uterus in position. Among them, a "ligamentum cardin-
ale uteri" (transverse cervical ligament) is known. Studies
of a plastinated series of slices from the female pelvis failed
to reveal collagenous fibers in the position corresponding
to these ligaments (Br6kelmann 1986). These problems have
hitherto been approached by dissection or by reconstruction
from histological sections, Dissection of tiny "filaments"
structures is difficult and structure may be misinterpreted
as a result of changes caused during the dissection proce-
dure itself, whilst reconstruction from a series of histologi-
cal sections is limited to relatively small areas. One of the
main advantages of transparent body slices is, that the ar-
rangement of all tissue components can be studied in their
undisturbed context.
Figures 13 and 14 illustrate an embryological research
application dealing with the formation of connective tissue
boundaries in the subperitoneal space of the fetal pelvic
cavity. In the retro- and pararectal regions of the fetus,
there is a progressive condensation of connective tissue la-
mellae. Within preexisting mesenchymal spaces, fat lobules
start spreading. In fetuses between 10 and 20 weeks, speci-
mens plasfinated with epoxy resin (E t2) and cut on a dia-
mond wire saw into 300-700 ym thick sections offer the
best opportunities for architectural studies (Fritsch 1986).
Such specimens fill the gap between paraffin serial sections
(suitable for younger embryos) and dissections of larger
fetuses. Paraffin sections of decalcified specimens in this
age group (10 20 weeks) exhibit separations between bone
or cartilage and the adjacent connective tissue due to
shrinkage and cutting problems. A better understanding
of this compartmentation is of clinical interest with respect
to the spread of infections and tumors, and to surgical ac-
cess.
Pathological alterations of connective tissue architecture
is another area of interest (e.g., in connective tissue diseases
such as polyarthritis). An investigation of the disturbed pat-
tern of the collagenous strands and fascia in Dupuytren's
contracture (Meinel 1983) has proved the applicability of
plastination in this field. Meinel found no shortening of
Fig. 9. Horizontal body slice, 2,5 mm thick, of a male pelvic regmn, processed by sheet plastination with epoxy resin (E 12), unstained.
The bones shown are the symphysis, tuber ischiadicum, and the neck region of the femur
Fig. 10. A close up of an adjacent large slice, approximately life size. It shows the pubic symphysis with adductor muscles, the internal
obturator muscle (left) and lhe prostatic gland and rectum, both encircled by the levator ani muscle
Fig. 11. Horizontal brain slice, processed by sheet plastination of a brain slice with polyester copolymer (P 35); 4 mm thick, unstained
Fig. 12. A close-up of the sheet of Fig. 11, revealing the detailed pattern of the basal ganglia and the anterior commissure
415
preexisting collagenous strands but rather a proliferation
of fibromatous tissue.
With respect to biomechanical problems sheet plastina-
tion offers an unique opportunity to study simultaneously
all components of a particular functional unit (muscles, col-
tagenous structures, cartilage and undecalcified bone). In
large specimens (longitudinal slices through the entire verte-
bral column), bones, discs and ligaments can be analyzed
in any flexed or extended position (Pfeil 1986). Together
with the possibility of studying the microangiographic pat-
tern in the same specimen (after filling with contrasting
resin), this technique appears to be a powerful tool in exper-
imental morphoIogy, especially in biomechanical and exper-
imental orthopedic research.
Another field for research applications concerns the
growth of tumors and the distribution of pathological tis-
sues. There are still major deficiencies in our knowledge
of the spread of malignancies within a particular area such
as the pelvis or the extraperitoneal cavity of the abdomen,
or about the spread of metastases to a remote area. In
a series of plastinated body slices, the distribution of malig-
nancies is immediately apparent to the naked eye - includ-
ing the possibility of checking the diagnosis by histological
samples. Use of this technique has also been made in sur-
gery. A complete screening of tissue amputates (e.g., female
breast, lung) for metastases is easily and completely
achieved by the combination of plastination and histology
(Guhr 1986). The results are important for both prognosis
and treatment. With respect to breast cancer, this time and
labor saving technique will probably be introduced as a
routine staging tool.
For subsequent histological investigations, PEM-im-
pregnated specimens are also of special value. Areas of in-
terest are localized by the use of computer tomographs
which are taken from the cured whole specimens (Klemstein
4985).
A last example of applied plastination techniques is in
anatomical research in individuals of rare species. It is obvi-
ous that plastination of an entire organism as a complete
series of transparent sheets allows structural investigations
at successive levels of resolution, from gross anatomy down
to the level of electron microscopy, provided, that an ade-
quate fixation has been applied (Klemstein 1982). Thus,
the cadaver of a rare animal (e.g., a whale embryo) could
be preserved, studied and kept in stock for future investiga-
[ions.
Principle of the plastination procedure
Fixation and dehydration
Most plastinated specimens start from fixed material and
every established fixation method is applicable. We use for-
416

Fig. 13. Transverse section, 600 ym thick, of the pelvis of a male fetus, crown-rump length /83 mm. Diamond wire saw section from
epoxy plastinated (E 12) specimen, stained with azur II-methylene blue and basic fuchsin as counterstain, x 4.5. Courtesy of Dr. Helga
Fritsch, Anatomisches Institut, Universitfit Bonn

Fig. 14. A close-up of the specimen of Fig. 13, revealing purple connective tissue lamellae between the pelvic bones and the rectum
preserved in their natural, undisturbed position, x 16

malin in concentrations between 5 and 20%. To enhance
color preservation, the Kaiserling solution (Kaiserling 1895)
is suitable (Fig. 5). Perfusion-fixed animals and arterially
injected human cadavers yield well fixed organs in their
correct anatomical positions. Organs removed at autopsy
are fixed by immersion and infiltration, taking care to re-
store their natural shape. Hollow organs have to be dilated
during fixation, a step essential in heart plastination (Tiede-
mann and v. Hagens 1982). All embalming fluids containing
long chain alcohols (e.g., glycerol) have to be removed be-
fore dehydration, as they easily spoil the final specimens.
Fixation can be omitted when epoxy resins are used (epoxy
resins have fixing properties), resulting in better color pres-
ervation. However, care should be taken with respect to
possible infections.
Dehydration and defatting are mandatory since water
and lipids can not be exchanged directly against polymers.
As known from histology, a proper dehydration procedure
must avoid shrinkage. The degree of defatting is essential
in plastination. The two methods used are stepwise dehy-
dration in graded ethanol and freeze substitution with ace-
tone.
Ethanol dehydration is used when a histological exami-
nation of the plastinated specimen is intended. The disad-
vantage of ethanol is its need for replacement by a low
boiling intermediary solvent (acetone or methylene chlo-
ride). In addition, stepwise ethanol dehydration causes con-
siderable shrinkage (roughly 50%, Schwab and v. Hagens
1981) and is time consuming. Ethanol dehydration is advan-
tageous for embalmed specimens, because it easily removes
(especially after addition of H202) the long chain alcohols
contained in the embalming fluid.
The standard dehydration procedure for plastination is
freeze substitution in acetone at - 2 5 ~ (Fig. 15). This
method saves time, requires less labor compared with etha-
nol dehydration, and causes only minor tissue shrinkage.
It is the only way to dehydrate brain tissue with a tolerable
(less than 10%) shrinkage (Schwab and v. Hagens 1981).
During freeze substitution, specimens (precooled to + 5~ C)
freeze immediately on immersion into the ice cold acetone
(technical grade), thereby stabilizing the specimens shape
instantly. Within the next 3-5 weeks (including 2-3 changes
of the acetone), the specimens become completely dehy-
drated. The only disadvantage of freeze substitution is the

/ Freezer,
-3-
Compressor
formation of ice crystals in specimens which will also be
used for histology. This can be overcome by processing
specimens containing 20-100% formalin which acts as a
freeze protecting agent.
In ethanol dehydration, the defatting is accomplished
simultaneously with the dehydration procedure. In freeze
substitution, defatting requires an additional acetone bath
at room temperature. For lipid-rich specimens (containing
bones, subcutaneous or subserous adipose tissue), defatting
may be achieved in a final bath of methylene chloride. For
plastination purposes, acetone is ideal because it acts as
dehydration agent, defatting agent and intermediary solvent
all at the same time and readily mixes with all the different
resins used for plastination.
Forced impregnation
The central and most important step in plastination is re-
placement of the intermediary solvent (which occupies the
spaces originally filled with water and lipids) by curable
polymers. This is achieved by means of a vacuum during
forced impregnation. The specimen, soaked within a vola-
tile intermediary solvent (acetone or methylene chloride),
is placed into the polymer solution. The intermediary sol-
vent has a high vapor pressure and a low boiling point
(acetone: + 56 ~ C, methylene chloride: + 40 ~ C), while the
polymer solution has a low vapor pressure and a high boil-
ing point. Therefore, when a vacuum is applied, only the
intermediary solvent is continuously extracted out of the
specimen and through the surrounding polymer solution
in the form of gaseous bubbles. The surrounding resin is
also eventually cleared completely of the intermediary sol-
vent, and therefore only one impregnation bath is needed.
Thus, the quantity of polymer consumed is very small and
approximates, for silicone, at most to the volume of the
specimens.
The speed of impregnation depends both, on the speci-
men and on the class of polymer used. Generally, polymers
with a higher viscosity require a longer impregnation time
than polymers of lower viscosity. The larger and denser
the specimen, the slower an impregnation should be pre-
ferably with a low viscosity resin. To accomplish impregna-
tion, any small oil driven vacuum vane pump is sufficient,
speed must be adjusted by varying the vacuum by means
of an air bypass valve. Gas bubble formation, easily ob-
served through a window, is a useful indicator of the speed
of impregnation. A too rapid extraction of the intermediary
417
at-25~ /
Fig. 15. Set-up of equipment for
dehydration by freeze-substitution
and for vacuum-impregnation as
used in the standard silicone
plastination process. As a safety
precaution, the compressor of the
freezer and the vacuum pump have
been removed and placed in an
adjacent room
solvent causes a collapse of the structural framework of
the specimens (i.e., shrinkage) and must be prevented. Very
delicate specimens are preferably processed via methylene
chloride. Its high specific gravity (1.3) ensures that the speci-
mens will sink to the bottom of the impregnation bath (spe-
cific gravity about 1) and therefore they must not be
weighted down.
Forced impregnation is carried out either at room tem-
perature when using epoxy and polyester resin or at - 25 ~ C
in a deep freezer when working with silicone rubber. In
the cold, the gas bubbles will rise slowly to the surface,
without splashing. A final vacuum in the range between
2 and 15 mm Hg is necessary. At room temperature forced
impregnation generally goes faster and with strong bub-
bling, since the polymers are less viscous and have a shorter
processing time.
Curing (hardening)
The curing of the specimens is carried out after removal
from the impregnation bath; the residual polymer of the
bath should stay fluid for re-use. Three specific techniques
are applied, which differ markedly from the usual curing
of polymers cast in molds.
A gas curing procedure, specially developed for plastina-
tion is used for silicone specimens (v. Hagens 1982). In
this technique, the decisive crosslinking curing agent is not
a constituent of the impregnation bath, but is applied later
to the specimens in a gaseous form. In a closed chamber,
the impregnated specimens are exposed to an atmosphere
which is continuously saturated with the gaseous hardener
evaporating from a stock solution inside the chamber.
Continuous evaporation and circulation of the gas is
achieved by a small membrane pump speeding up the curing
process.
The curing of specimens impregnated with epoxy resin
(E 12) or polymerizing emulsion (PEM) takes advantage
of the tissue amines contained within the specimens. Amines
are effective accelerators. Together with anhydrides, used
as hardeners for epoxy resins or PEM impregnation mix-
tures, they are sufficient to fully cure the specimens at
+ 50 ~ C.
Curing of polyester-copolymers can be initiated by
UVA-light followed by a heat treatment ( + 50 ~ C). The in-
herent advantages are a long manfacturing time in the dark,
and a lower temperature peak during their markedly exo-
thermic curing.
418
Table 1. Standard silicone plastination (S 10)
General procedure Weeks
Fix with formalin, dissect. 1 in the specimens framework, resulting in a white appear-
Optional: Macroscopic staining, or vascular injection
prior to fixation
Dehydrate by freeze-substitution in acetone 1-5
(-25 ~ C) until water content is below 1%
Impregnate with BiodurTM S 10/S 3 in vacuum 3 A specimen plastinated with Polymerizing EMulsion
( - 25~ C)
Remove from silicone solution, drain and wipe off 3 technique is especially suitable for thick body slices
and harden by gas curing process
Total time: up to i2 weeks
Equipment required: Impregnation unit (vacuum container, vacu-
um pump, manometer, bypass valve), deep freezer (Fig. 15)
Plastination techniques
Standard silicone technique (Table 1)
The S 10 standard silicone technique (other silicones being
limited to special purposes; v. Hagens 1985/86) produces
flexible, resilient and opaque specimens (Figs. 1-5). After
fixation and dehydration, the specimens are impregnated
at - 2 5 ~ with a silicone base material (S 10), containing
a thickener (hardener S 3). At this low temperature, the
reaction mixture remains sufficiently fluid for at least three
months. It can be used up during subsequent cycles. Speci-
mens covered with skin or dense capsules (e.g., limbs, fe-
tuses, testes), which act as a barrier to silicone, require a
supportive infiltration with the impregnation mixture; oth-
erwise they will shrink.
After impregnation, the specimens are cured by a silicate
containing gas, evaporating from a fluid (S 6). The hardener
S 3 contained in the impregnation bath initiates the curing
of the silicone molecules by end to end polymerization.
Due to crosslinking during the final gas curing, the silicone
rubber within the specimen will become solid and dry. Dur-
ing the curing process, the specimen's surface can be ob-
served and easily cleaned. The surface cures within hours,
but diffusion of the gas into the center of the specimen
is slow. To ensure proper curing throughout, the specimen
has to be bagged for a couple of weeks, allowing a uniform
distribution of the gas curing agent within the specimen.
As room temperature speeds up the thickening of the sili-
cone base by the hardener S 3, less and less gas curing
agent S 6 is required for crosslinking the silicone through-
out the specimen.
Cured specimens can be ground or carved, but dissec-
tion proper is impossible because the cured specimens pos-
sess uniform firmness. The initial dissection should there-
fore be thorough.
A variation of the silicone standard technique is used for
plastination of thin-walled specimens (e.g., intestine) or
spongy specimens (e.g., lung slices). This is intended to keep
even inner surfaces virtually free of polymer.
Central to this technique is the addition of a high boiling
solvent to the reaction mixture which dilutes the polymer
(e.g., xylene). During forced impregnation, this solvent is
essentially not extracted. During curing it is not allowed
to evaporate before sufficient hardening of the specimens
has been achieved. Shrinkage due to solvent evaporation
is thus avoided. Consequently, the specimen is impregnated
with a reduced amount of resin. Because the added solvent
is replaced by air during evaporation, the resin accumulates
ance.
Polymerizing emulsion technique
( = P E M ) is opaque and firm, but not unbreakable, This
(15-30 mm) and for showcase specimens (Figs. 6 8). Opa-
queness is not easily obtained. As described by Beyer (1908)
and by Spalteholz (1914), a specimen becomes transparent,
when the refractive index of the imbuing medium matches
that of biological tissue (nD20 = 1,56). This holds true only
for defatted specimens. Therefore, dehydrated and defatted
thin tissue slices plastinated with epoxy or polyester resins
(nD20 = 1,52 -- 1,58) become transparent to a varying extent.
Thick slices may become dark due to a high blood content
and/or superimposion of structures. Only specimens plas-
tinated with silicone rubber (nD20=l,40) are always
opaque.
In the absence of a thermosetting resin with a suitable
refractive index to yield opaque specimens, emulsifying
epoxy resins have been introduced (v. Hagens 1979b). The
formula stays homeogeneous and clear during impregna-
tion (Fig. 7), but emulsifies during curing (Fig. 8). The
cured emulsions are composed of a continuous epoxy phase
and a discontinuous silicone phase. As with frosted glass,
light transmittance is combined with light dispersion, and
the final specimen, impregnated with this polymerized
emulsion, is opaque.
PEM-polymers have the practical advantage, that
forced impregnation can be carried out at room tempera-
ture. The resin can be used for several weeks, if curing
is accelerated by tissue amines. Thus, curing during forced
impregnation takes place only within the specimens, and
not within the surrounding PEM-polymer bath.
Sheet plastination technique
Sheet plastination refers to the preservation of large (up
to 50 x 25 cm) and thin (down to 2 ram) slices of organs
and whole bodies. In this technique, the water and part
of the lipids are replaced by thermosetting resins, namely
epoxide or polyester resins. After fixation, dehydration and
forced impregnation, the slices are cured between polyester
foils, tempered glass plates, or glass plates covered with
polyester foils. There are two methods of sheet plastination,
the "draining" and the "filling method".
The draining method (Table 2) is simple and very useful
in combined macro-histological studies. In short, the im-
pregnated tissue slices are put between polyester foils,
avoiding entrapment of air bubbles. The sheets are covered
with glass plates and clamped together. This "sandwich"
is put into a slightly oblique position, allowing surplus resin
to drain off from around the slices. If many slices are to
be plastinated, as many as 40 layers of slices can be piled
up between 41 glass plates and polyester foils.
Use of such slices is advantageous in combined macro-
histological studies because they can be studied in their
topographical context from the macroscopic down to the
electon microscopic level. Plastinated tissue slices can be

Table 2. Sheet plastination of transparent slices, draining method
with epoxy resin (E 12)
Cut fixed tissue with a rotary meat slicer into h
about 2.5 mm thick slices.
Stain nuclei (hemalum) or connective tissue h
(modified Azan stain)
Dehydrate in graded ethanol and transfer to 1 day
acetone as intermediary solvent
Impregnate with epoxy resin (Biodur TM E 12) 1 day
Place slices between polyester foil and/or glass h
plates and clamp together
Cure at room temperature and/or at + 50~ C 1 day
in slightly obliqe position
Remove polyester foils and glass plates
Total time: 3-5 days
Equipment required: Impregnation unit (vacuum container, vacu-
um pump, manometer, bypass valve), deep freezer (Fig. 15), meat
slicer, band saw for bone-containing specimens
Table 3. Sheet plastination of transparent slices, filling method with
epoxy resin (E 12)
Freeze unfixed or fixed specimens 2 days
Saw into 2.5 mm slices 1 day
Stack between plastic nettings and grids, h the size of the glass plates and the thickness of the flexible
avoid thawing and drying
Dehydrate by freeze substitution with acetone 1 week
followed by defatting in methylene chloride
Immerse in E 12/E 1-reaction mixture h by vacuum, and curing is accomplished by UVA-light and/
Impregnate in vacuum to under 2 mm Hg 1 day
Place slices onto glass plate, assemble flat h one for transparent body slices, using epoxy resin, the other
chamber and fill
Cure at room temperature or at + 50~ C 1 day
Total time: 2 weeks
Equipment required : Large vacuum chamber (also for the removal
of air bubbles), manometer, bypass valve, deep freezer, band saw.
Auxiliaries : glass plates, gaskets, clamps, plastic grids, nettings and
grids.
studied directly under a binocular dissecting microscope,
using combined transmitted and reflected light. Contrast
can be enhanced by applying modified staining procedures
(e.g., diluted, prolonged Azan staining) before dehydration.
For screening of malignancies in amputated tissue such as
lung and breast, staining of the nuclei (e.g., with hemalum)
has proved suitable (Guhr 1986). Tissue areas of special
interest can be cut out, mounted and processed for light
and electron microscopy. Microscopic sections can not only
be stained with routine plastic staining procedures (e.g.,
Richardson's stain), but also with H.E., a procedure pre-
viously applied only to acrylic sections.
The filling method (Table 3) produces plastinated tissue
slices of uniform thickness with smooth, glass like surfaces
(v. Hagens 1977, US-Pat. 4, 320, 157, 1982).
Central to this method is the assembly of a flat chamber
around the impregnated tissue slice. This chamber consists
of two tempered glass plates, separated from each other
by a flexible, elastic gasket. Its volume is determined by
419
Fig. 16. Set-up for sheet plastination showing a flat chamber com-
posed of glass plates, separated by a flexible gasket and held to-
gether by clamps. A slice is sandwiched between the glass plates,
and resin fills the chamber
gasket (Fig. 16). Within the flat chamber, the impregnated
tissue slices are held between the glass plates. Subsequently,
the flat chamber is filled with resin, air bubbles are removed
or heat. There are two main variations of this technique,
for brain slices, working with polyester-copolymer.
Transparent slices of bodies, organs or extremities are
produced with the E 12-technique. The optimal thickness
is 2.5 ram; thinner slices are too pale, while in thicker slices
too many structures may be superimposed. Transparency
results from lipid extraction and because the refractive in-
dex of the cured resin matches that o f the tissue (nD20 =
1.56). The specimens are cut in the frozen state with a band
saw. Slices larger than 10 cm need to be cut on a sliding
table with a cooled guide stop (0 ~ C). After dehydration
by freeze substitution in acetone and defatting in acetone
or methylene chloride at room temperature, the tissue slices
are then impregnated in a mixture of an epoxy resin with
the appropriate hardener. For curing, the slices are pro-
cessed by using the flat chamber assembly mentioned above.
Nontransparent brain slices, cast as sheets (Figs. 11, 12),
are produced by the P 35 technique (Table 4). Fixed brains
are cut on a meat slicer, 4~-6 m m thick. The single slices,
supported by wet filter paper on one side, are stacked in
a rack of stainless steel flow-through trays. As brain slices
become very brittle during freeze substition in acetone, they
are processed in this cage until casting.
The resin used for brain slices (P 35) is a transparent
polyester copolymer, cured with a light sensitive peroxide
hardener (A 9). The cured brain slices are suspended in
sheets. The differentiation between gray and white matter
surpasses any macroscopic staining method such as Prus-
sian blue. The cured slices (Figs. 11, 12) are opaque because
of the preservation of tissue lipids. This is achieved by short
freeze substitution dehydration (acetone at - 25 ~ C), avoid-
420

Table 4. Sheet plastination of brain slices with polyester resin (P 35)
Cut formalin-fixed brains into 4 mm-slices
and stack on flow-through trays
Rinse and cool to + 5~ C in deionized water
Dehydrate by freeze-substitution in acetone
at - 2 5 ~ C
Impregnate with polyester resin Biodur TM (P 35)
under vacuum (darkened)
Assemble flat chamber (Fig. 16), fill up,
and cure under UVA-light
Post-cure at + 50~ C
The resulting reddish tint renders the specimens somewhat
more lifelike.
h
Other applications
16 h
2 days Plastination is not confined to the field of the morphologi-
cal sciences. Archeologists have started to use plastination
1 day for the preservation of textile structures that can not be
dried, such as leather, rope and wood. Even bread and
cake have been plastinated. Insects stay flexible and are
45 rain
more durable than after simple drying. Stained transparent
slices also find interest as pieces of natural art.
5 days

Total about 10 days Feasibility

Equipment required: Impregnation unit (vacuum container, vacu-
um pump, manometer, bypass valve), meat slicer, 2 UVA-lamps,
2 fans. The plastination of a single brain slice requires 4 tempered
glass plates, 35 clamps, one flexible gasket, and two stainless steel
grids.
ing major lipid extraction. Forced impregnation is carried
out in a darkened vacuum chamber at room temperature.
The vacuum must not fall below 10 m m Hg, otherwise the
styrene of the impregnation bath will be extracted and spoil
the oil of the vacuum pump.
The curing process is highly exothermic; it is initiated
with UVA-light at room temperature, followed by oven
curing at + 45 ~ C. Curing is accompanied by a 15% shrink-
age. Since this would cause annoying boundary lines ar-
ound the impregnated slice if casting were carried out be-
tween 2 tempered glass plates as for sheet plastination with
E 12 (filling method), brain slices are cast in double-walled
glass plate chambers. The elasticity of the thin, inner glass
plate prevents the formation of boundary lines, whilst an
outer security glass plate gives firm support.
Auxiliary techniques
Vascular injection
Before fixation, the vasculature of organs or limbs can be
filled with colored epoxy resins. If desired, filling can be
achieved down to the capillary level. This increases the in-
formational value of specimens such as kidney, placenta
or heart (Fig. 3). A specially formulated and colored epoxy
resin (Biodur TM E 20) is used for injection. It has good flow
properties on wet surfaces (e.g., endothelium), cures well
in the presence of tissue with less than 2% shrinkage, and
does not break down in acetone (in contrast to acrylates
as used for corrosion casts). For the injection of delicate
organs, such as gut, the resin E 20 should be diluted with
a solvent or a plasticizer, thereby reducing its viscosity and
prolonging its working life. Another additive for an op-
tional x-ray contrast is an organic iodine compound.
Bleaching and staining
After fixation, bleaching of specimens (e.g., joints, intestine,
genital organs) by H202 (2-5% of the commercial HzO 2
solution in 5% formalin) is recommended. A specific stain
for specimens to be plastinated has been formulated (Bio-
dur TM CR). It can be dissolved in a formalin bath or simply
added to the acetone bath during dehydration (specimen
of Fig. 2). Before staining, specimens must be bleached.
What are the practical requirements for a plastination labo-
ratory with regard to expense, time expenditure and opera-
tor experience? Basically, this technique is no more de-
manding than the processing of tissue for paraffin histology
or electron microscopy. For the silicone S 10 and PEM-
techniques, the apparatus required is neither very costly
nor very sophisticated, being cheaper than a freeze drying
apparatus. The cost of materials needed is under US $ 40
per kg of plastinated tissue and can be minimized when
all the resin is used up in continuous runs of vacuum im-
pregnation. It is therefore advisable always to have a
number of dehydrated specimens in stock. Change of sol-
vents, staining, vacuum surveillance, as well as the time-
consuming aftercare during the curing process, require at
least a half time commitment for one person. One should
not start a routine plastination laboratory with less avail-
able time than this. Nevertheless, during plastination, there
are several stages at which the process can be brought to
a long time halt. This is possible for fresh specimens in
a deep freezer or later in 70% ethanol or in methylene
chloride.
Sheet plastination, except for the draining method, is
more demanding, expecially when whole body slices are
to be produced. The manufacture of more than just a few
sheets per month requires a significant number of safety
glass plates (e.g., four glass plates per brain slice), a washing
machine, various dehydration baskets and grids (in order
to stack the tissue during dehydration), large dehydration
containers, ovens and vacuum chambers. If using the drain-
ing method, the chemicals for a slice 10 x 10 cm cost ap-
proximately US $ 5. A trunk sheet, processed by the filling
method requires approximately US $ 40 worth of chemicals,
a brain slice approximately US $ 25. For larger (trunk)
or harder (head) slices, a precise, sharp band saw is essen-
tial, equipped with a sliding table and a sliding guide stop
that must be cooled. A deep freezer at - 7 0 ~ C is desirable
to avoid thawing of the specimen during sawing. The frozen
saw dust on the cut surfaces has to be scraped off by the
use o f an open (air circulated) deep freezer. Thus, a realistic
estimate for the establishment of a sheet plastination labo-
ratory employing the filling method approximates to US
$ 50 000 for equipment. This is double the price of an ultra-
microtome, and thus within the reach of most anatomical
institutes.
Plastination in comparison with polyethylene
glycol preservation
Alternative methods for the preservation of handable speci-
mens are limited. Besides paraffinization, impregnation

with polyethylene glycols is o f importance. These exist as
a hygroscopic liquid ( P E G 200~600) a n d as a solid with
the consistency o f bees wax ( P E G 1000 and higher). P E G s
are not inflammable. A great advantage is their water solu-
bility which means that fixed specimens can be impregnated
by simple immersion without any dehydration. Liquid
P E G s yield the best moist specimens that can be produced
at the m o m e n t ( G e y m a y e r and Gfitebier 1979). It is to be
noted that they lose their formalin o d o r due to the forma-
tion of hemiacetales. This m e t h o d is recommended for or-
gans and joints to be used in the dissection lab. Impregna-
tion can be speeded up by vacuum ( G e y m a y e r and Gfitebier
1979; Kienecker and U h l m a n n 1985 a, b).
Only dry PEG-specimens from solid P E G s can be com-
pared with plastinated specimens. As with paraffinized
specimens they are brittle and dark, exhibiting less detail
than the m o r e durable and lifelike plastinated specimens.
Acknowledgement. In the development of polymers for plastination,
we want to acknowledge the cooperation and advice of Dr. Karl-
Heinz Rudolph, Bayer AG, Leverkusen, Dr. Wolfgang Koser,
BASF, Ludwigshafen and Dr. Gerhard Stenbeck, Bakelite GmbH,
Duisburg.
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