Journal of Anatomy

J. Anat. (2017) 231, pp309--317 doi: 10.1111/joa.12634

Saturated salt solution: a further step to a

formaldehyde-free embalming method for veterinary
gross anatomy
M. Lombardero,1 M. M. Yllera,1 A. Costa-e-Silva,2 M. J. Oliveira2 and P. G. Ferreira2
1
Department of Anatomy, Animal Production and Clinical Veterinary Sciences, Faculty of Veterinary Sciences, University of
Santiago de Compostela - Campus of Lugo, Lugo, Spain
2
Department of Anatomy and Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel
Salazar (ICBAS), University of Porto, Porto, Portugal

Abstract
In the field of veterinary anatomy, most of the specimens used in practical sessions are perfused with fixatives.
Thus, they can be used for a longer time, reducing the number of animals for educational purposes. Formalin is
the most commonly used fixative, consisting of a 37% formaldehyde solution. However, formaldehyde is a
powerful irritant of the eyes and airways and is considered carcinogenic, causing nasopharyngeal cancer in
exposed workers and professionals. In the present study, we explored an alternative method to avoid the use
of formaldehyde in specimens used for gross anatomy practical sessions. We propose an inexpensive, non-toxic
fixative that is available worldwide, such as sea salt. This method consists of a continuous perfusion of
saturated salt solution for a period of 6–8 h, enabling drainage of the solution to avoid a weight increase of
the specimen, and allowing salt to be retained in the tissue. The method is based on recirculation of the
saturated salt solution instead of maceration. Perfused specimens retained their natural consistency and joint
mobility, with no blood, resembling a piece of meat from the slaughterhouse. They could be used immediately
without a maceration period, or stored in the fridge until use and then kept in a bath of saturated salt
solution for future conservation. In the case of the former, no refrigeration was needed. The specimens did not
have an irritating or offensive smell, and could be used for long sessions (several hours per day) and stored for
long periods. However, the blood vessels used for perfusion determine the results: a less invasive approach
(through common carotid arteries) gave good preservation of the musculoskeletal system, whereas more
invasive access to cannulate the abdominal aorta and vena cava caudalis was required to achieve better
preservation of the viscera. In conclusion, we propose that perfusion followed by immersion in a saturated salt
solution is a good alternative method for the preservation of specimens used in the practical teaching of gross
veterinary anatomy. It is a very simple and inexpensive technique, and is much healthier for users than
traditional formalin. Moreover, specimens can be preserved for prolonged periods, and maintain a similar
appearance and consistency to fresh material.
Key words: conservation; dissection; education; formalin; gross anatomy; salt solution; veterinary anatomy.

animal. However, a fresh specimen can only be preserved

Introduction
When studying anatomy, working on a fresh specimen is
ideal because its consistency is similar to that of a living
Correspondence
Matilde Lombardero, Department of Anatomy, Animal Production
and Clinical Veterinary Sciences, Faculty of Veterinary Sciences,
University of Santiago de Compostela - Campus of Lugo, 27002 Lugo,
Spain. E: matilde.lombardero@usc.es
Accepted for publication 6 April 2017
Article published online 24 May 2017
for an extremely short time. Many attempts to preserve and
embalm corpses and animal specimens have been made
since ancient times in order to retain the body’s morphol-
ogy after death. Different periods are well-described by
Brenner (2014) leading to modern anatomical preservation.
Initially, people used alcoholic solutions of arsenic and/or
alumina salts in different concentrations (Brenner, 2014).
Later on, carbolic acid and phenol were incorporated into
embalming solutions until formaldehyde was discovered as
a preservative at the end of the 19th century. Since then,
formalin has been extensively used in medical schools for
anatomical embalming. Although it is considered a

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310 Removing formaldehyde from veterinary gross anatomy, M. Lombardero et al.
universal fixative, it is far from perfect, as it hardens and
dehydrates the tissues and gives them a grey hue, which
does not provide a very realistic model. In addition, it coag-
ulates the blood and has an unpleasant smell (Brenner,
2014; Hayashi et al. 2016).
Formaldehyde users suffer from skin irritation, burning
eyes, headache, irritation of the respiratory system and,
after prolonged inhalation, dizziness and epistaxis (Wantke
et al. 2000; Brenner, 2014). These symptoms are more sev-
ere depending on concentration degree and exposure time.
Formaldehyde is particularly dangerous as, in addition to its
irritant (eye irritation in man occurs in concentrations
between 0.1 and 1 ppm) and allergenic (skin sensitisation)
actions, it is classified as a carcinogenic substance. The
World Health Organization recommends, as an air quality
value, to not exceed 100 lg m 3 for an average time of
30 min to avoid complaints from sensitive people due to
irritation and odour. In 2006, the International Agency for
Research on Cancer (IARC) included formaldehyde in Group
1 (carcinogenic to humans) because there is sufficient evi-
dence that formaldehyde is carcinogenic in both animals
and humans, causing nasopharyngeal cancer in workers
and professionals exposed to it (such as anatomists and
pathologists), as well as leukaemia (myeloid type) (IARC,
2006).
In an attempt to reduce its adverse effects on exposed
teaching staff and anatomy students, several modified
formulae have been published in the scientific literature.
Among the latest methods, Thiel’s embalming technique
conserves the whole corpse, with a texture and colour
close to that of living tissue (Thiel, 1992a,b); however, it
is a complex procedure and uses various reagents, some
of which are also noxious. Plastination (Von Hagens,
1982) is another example of body embalming, and is very
useful for teaching gross anatomy, but it is not suitable
for dissection practice or surgical training. Unfortunately,
it requires a very complex and expensive procedure,
which is not suitable for everyone because of its high
cost. Phenoxyethanol was also used to wash out excessive
formalin and attenuate its odour from cadavers (Rumph
& Williams, 1988). Other authors have used modified Lars-
sen solution (Silva et al. 2007) in an attempt to reduce
formaldehyde use, resulting in dog cadavers with colour,
tissue texture, skin and joint flexibility quite close to that
of living dogs, although formaldehyde is still present in
its formula.
Janczyk et al. (2011) used nitrite salt as an alternative for
embalming. They compared this new method with
formaldehyde fixation, initially with isolated limbs from
dogs. Later on they used the whole animal and also
removed ascorbic acid from their formula because of the
yellowish colour of the specimens. In addition, they
reported they had to open the abdominal cavity to facili-
tate the perfusion of viscera by the fixative when specimens
were immersed in tanks.
Embalming of human corpses with a saturated salt solu-
tion has been widely tested and accepted for medical train-
ing, especially for surgical skills training (Hayashi et al.
2014), as well as plastic surgery (Shirai et al. 2015). These
authors used the same formula: a solution made up of satu-
rated sodium chloride, 20% formaldehyde, phenol, glycer-
ine, isopropyl alcohol and water. However, although it
gave a realistic model, the solution reported by Hayashi
et al. (2014) still included some hazardous compounds such
as formaldehyde and phenol.
After many years of exposure to formaldehyde, sensiti-
sation increases irritation. In addition, the European Asso-
ciation of Veterinary Education (EAEVE) recommends
replacing this fixation method by a non-toxic alternative
suitable for preserving anatomical specimens (EAEVE,
2010), and advises using the preservation method of Fri-
ker et al. (2007). That method consists of brine made up
of 87% tapwater and 13% pickle salt, with some non-
specified antioxidants, and it can be used in two ways:
perfusion or immersion. As our dissection facilities have
no devices to reduce or remove formaldehyde fumes, stu-
dents and teachers of anatomy are subjected to haz-
ardous doses of formaldehyde. We have therefore
explored an alternative method (inspired by Friker’s) to
avoid the use of formaldehyde in animal specimens used
for gross anatomy practical sessions. Our practical sessions
last for more than 2 weeks during the first semester and
another 2 weeks in the second semester, during which
time the bodies are kept at room temperature for at
least 6–7 h a day. This prevents the use of fresh or frozen
animal corpses. The aim of this study was to avoid the
use of formaldehyde-fixed material during gross anatomy
practical sessions in an effort to improve our dissection
room environment by the use of an alternative, simple
and inexpensive method, based on a widely available
and non-toxic compound, such as sea salt.
Materials and methods
Only two common compounds were used: salt (commercial NaCl,
sodium chloride) for human use and tapwater. Two types of salt
solution were prepared: salt solution at 0.9% (SS) for washing and
removing blood from blood vessels, and saturated salt solution
(SSS) for perfusion and bathing. A saturation point of NaCl of
35.89 g 100 mL–1 of water at 20 °C was achieved, taking into
account that solubility increases with temperature (38.99 g salt dis-
solves in 100 mL of water at 100 °C) (Green & Perry, 2008). The salt
solutions were filtered before perfusion to remove impurities, by
gravity or simple filtration, using regular filter paper placed in a
funnel. For the immersion tanks, SSS does not need to be filtered,
and it is advisable for some excess salt to remain in the brine.
Cadavers of dogs were obtained from the City Pound where the
animals were sacrificed by a euthanasia compound that also con-
tains heparin or an anticoagulant drug to prevent blood clot forma-
tion; this was done according to Regulation (EC) no. 1069/2009 and
was approved by the appropriate local ethics committee. The
preservation technique consists of perfusing a body with SSS,
© 2017 Anatomical Society
 Removing formaldehyde from veterinary gross anatomy, M. Lombardero et al. 311

replacing the blood in vessels with this solution. This process can be (e.g. 1–1.5 L for small specimens such as rabbit). After the
summarised in the following steps: initial drainage of the SS/SSS mixture, the further pure SSS
drain-out is collected in a container for recirculation. The
(1) Sacrifice animals with a euthanasia compound containing
entire SSS perfusion volume is injected before starting the
heparin or any anticoagulant drug to prevent blood clot
recirculation process.
formation.
(7) For the recirculation process, the pump’s feeding tube is
(2) Dissect large vessels of the neck (arteria carotis communis
immersed in the recirculated SSS fluid container, with an
and vena jugularis externa) bilaterally, as well as the vena
entry filter (regular filter paper) to prevent impurities from
femoralis. This must be performed before bleeding to facili-
clotting the vessels. The recirculation, body mass-depen-
tate the identification of vessels and subsequent cannula-
dent, is continued for 6–8 h for an animal with an average
tion.
body mass of 20 kg (a longer time is required to perfuse a
(3) Bleed animals through the common carotid arteries, prefer-
heavier animal). The perfusion process should be thor-
ably with the animal suspended from the hind limbs.
oughly surveyed, as the key point is that the outflow must
(4) Cannulate the previously dissected large vessels in both
be equal to the inflow. Thus, special attention should be
directions (i.e. cranially and caudally): arteries for perfusion
paid to the outlet points, checking that there is a continu-
and veins to drain the salt solution.
ous flow at all times. Otherwise, outlet cannulas should be
(5) Perfuse the washing solution (SS) using a peristaltic pump,
readjusted in place and/or the animal position has to be
to remove all of the blood until the fluid draining from the
changed. Moreover, the fluid level at the feeding/recycling
veins is clear in colour.
container should remain constant during the entire
(6) Perfuse the body with SSS. The SSS volume (L) is taken as a
procedure.
3 : 1 ratio to body mass (kg). Initially, the injected SSS will
drive out the SS remaining in the vessels. Therefore, the ini- The aim is to wash out the blood and recirculate the SSS for
tial outflow is discarded. The cut-off point to start recycling several hours, so tissues begin to incorporate salt from the
the SSS depends on the body mass. For guidance, it could inside, without any weight gain from the specimen by retaining
be set when reached an SSS volume of 1/3 of body mass SSS. Once the perfusion has finished, specimens can be used

A B

C D

Fig. 1 SSS perfused specimens. (A) Male dog

dissected by students 15 days after perfusion
displaying a very realistic image of the
muscles, nerves and vessels of the caudal part
of the abdomen and medial aspect of the
thigh. (B) Lateral view of the thigh and crural
regions from a male dog dissected by
students and stored in SSS for 18 months
showing no sign of degradation. (C) Female
rabbit showing good preservation of the
viscera. Image taken 16 months after
perfusion. (D) Transverse section of the
cranial part of the abdomen of a dog (caudal
view), formalin fixed, immersed in SSS and
displayed at the Museum of Anatomy at the
Abel Salazar Institute (University of Porto).

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312 Removing formaldehyde from veterinary gross anatomy, M. Lombardero et al.
immediately or kept at room temperature or in a refrigeration
chamber until dissection is performed. Once the dissection has
started, it is recommended to keep the specimens refrigerated
and, if used for longer than 1 week, immerse them in a bath
of SSS (no refrigeration is needed).
If the priority is the best preservation of thoracic and abdominal
viscera, access to the abdominal cavity is needed. After opening the
abdominal cavity, the aorta abdominalis and the vena cava caudalis
were sectioned caudal to the arteria mesenterica cranialis and cra-
nial to the arteria mesenterica caudalis. To obtain overall good per-
fusions, the cannulas that were inserted in the aorta or vena cava
were alternatively oriented to either the cranial and caudal side of
the artery or vein, thus allowing the perfusion liquid to run both
cranially and caudally.
Results
We tried two different ways of performing the embalming
process, and obtained different results depending on the
vessels that were cannulated:
(1) A less invasive approach, through cannulation of
the common carotid arteries as the entrance and
the external jugular and femoral veins as the exit,
enabled us to get good results for musculoskeletal
preservation (Fig. 1A–B), allowing for a wide range
of motion of the joints, similar to a living animal.
The exceptional results for the preservation of mus-
cles and joints indicate that this would be the
method of choice if the goal were a dissection of
the musculoskeletal system. In this approach, tho-
racic and abdominal viscera were not perfectly pre-
served. If cavities were accessed soon after SSS
perfusion (1 week), viscera showed a quite natural
aspect and consistency. However, after 6 months,
A B
the viscera had lost consistency and bright colours,
although they were not putrid or foetid.
(2) If the main interest is keeping the natural appear-
ance, colour and consistency of the thoracic and
abdominal viscera, an invasive approach is needed
and the SSS perfusion should be done through
abdominal large body vessels. For this, access to the
abdominal cavity is required to cannulate the aorta
abdominalis and the vena cava caudalis. This tech-
nique provided good preservation of the viscera
and the entire musculoskeletal system (Figs 1C and
2); no significant differences between cranial and
caudal halves of the animals with regard to organ
preservation were found. However, this approach
impairs the physical integrity of the cadaver, which
might not be suitable for a topographic approach.
Moreover, first year students may be apprehensive
when confronted with an open cavity with viscera,
and, as a matter of educational policy, students
are introduced to dissection starting with intact
specimens.
Perfused specimens maintained joint mobility and a
natural consistency (similar to fresh tissue, but without
blood), resembling a piece of meat from the slaughter-
house. Additionally, no unpleasant odours or irritating
vapours were generated. Tissues were easy to dissect,
as were their vessels and nerves, because they kept
their natural consistency, although vessel content was
clear. After SSS, the brain becomes harder than fresh
brain, which allows sections to be made in which
white and grey matter can be distinguished (Fig. 3).
The histological approach of some organs show that
Fig. 2 Male rabbit embalmed with SSS
through Aorta abdominalis and Vena cava
caudalis 5 years ago. It is the oldest specimen
kept at the Abel Salazar Institute (University
of Porto). (A) General appearance of the
specimen. (B) Major detail of the very well
preserved abdominal viscera, retaining their
natural appearance and texture.
© 2017 Anatomical Society
 Removing formaldehyde from veterinary gross anatomy, M. Lombardero et al. 313

their general organisation is usually preserved (Fig. 4),

although some lose their normal microstructure (e.g.
liver and spleen).
The proposed SSS method has other advantages: the
specimens could be used immediately after perfusion (no
maceration period was needed) and remained in good con-
dition regarding their consistency and appearance for a
medium to long period (during a whole course of two
semesters), as shown in Fig. 1B. Once specimens were
immersed in brine, they could be stored for a long time, 5
years at least (Fig. 2), with minimal maintenance. No evi-
dence of mould growth was observed when specimens
were stored individually in sterile containers or in vacuum.
Discussion
The present study proposes an alternative fixative method
for veterinary specimens to be used in gross anatomy teach-
ing. In this method, a saturated salt solution replaces com-
mon fixatives such as formaldehyde in the embalming
process. To use this preservation method, we conclude that
there are two main points, which are very important:
(1) Preventing blood coagulation, to allow a perfect
bleeding and the washing of the remaining blood
from the vessels with SS 0.9%;
(2) Embalming with recirculating SSS for a period of
time that is body mass-dependent.

Fig. 3 Macroscopic images of the encephala

of two dogs perfused 12 months (12 m) and
24 months (24 m) ago: (A) Encephalon in situ
(12 m) and removed from the cranium (24 m)
with patent gyri of neopalium. (B) Encephalon
removed from the cranium-dorsal aspect
(12 m) and ventral aspect showing the
hypophysis and the chiasma opticum,
suggesting that the tissue has sufficient
consistency (24 m). (C) Transversal section of
the hemisphaerium of the cerebrum in which
the white and grey matter could be clearly
distinguished. Manifest cutting marks might
be a sign of firmness. (D) Median section of
the metencephalon (pons and cerebellum)
where the difference between white and grey
matter is conspicuous.

© 2017 Anatomical Society

314 Removing formaldehyde from veterinary gross anatomy, M. Lombardero et al.

Regarding the first step, we believe that removing all of
the blood is essential to allow salt to reach the tissue. In
contrast, other authors (Hayashi et al. 2014) did not remove
blood from corpses before perfusing their SSS because their
formula was reported to liquefy the blood.
In reference to the second point, most embalming meth-
ods consist of perfusing a fixative or embalming fluid
through an artery while preventing any outflow, allowing
the fluid to fix body tissues during a maceration period.
Some authors infuse up to 25 L of fixative inside a human
corpse, as described by Hayashi et al. (2014), or even up to
8 Gal (30 L), as reported by Whitehead & Savoia (2008),
resulting in uniform swelling of the cadaver. In contrast,
Silva et al. (2007) perfused only up to 10% of the animal’s
bodyweight. Friker’s method is based on perfusion with
brine until it exits the nasal openings (Friker et al. 2007).
The specimen then needs to be refrigerated for about
1 week for fixation or maturation. Our technique is based
on the recirculation of SSS through the cannulated vessels
for 6–8 h depending on the animal’s weight. This is a com-
pletely innovative technique because continuous perfusion
has not been described by any authors in the literature. The
outflow should be equal to or balanced with the inflow to
avoid any accumulation of SSS in the body cavities or mus-
culoskeletal tissues, allowing the corpse to be embalmed
with salt from the core.

Fig. 4 Set of photomicrographs displaying some tissues and organs removed from two dogs perfused 12 months (first column) and 24 months
ago (second column). Sections were stained with haematoxylin-eosin. (A) Skeletal muscle tissue removed from M. temporalis. Peripheral nuclei and
cross-striations are still evident in muscle cells cut longitudinally. (B) Skeletal muscle tissue from the tongue preserving its general structure with
bundles of skeletal muscle cells oriented perpendicular to one another. (C) Cardiac muscle tissue satisfactorily preserved in the 12-month dog but
not as much at the 24-month specimen. (D) Nervous tissue from cerebellum displaying the molecular and granular layers of the grey matter and
the Purkinje cells. (E) Lung parenchyma is clearly identifiable in both specimens. (F) Kidney parenchyma hardly preserved in the 12-month specimen
but still recognisable in the 24-month specimen. (G) Hepatic parenchyma has completely lost its histological structure. (H) Splenic histology show-
ing stroma better preserved than parenchyma (with no sign of red and white pulp). In the 12-month dog, only the smooth muscle of the capsule
and trabecula is perceptible. Additionally, the reticular pattern is also visible in the 24-months dog. (I) Sections of testes showing a well preserved
organisation, with seminiferous tubules surrounded by interstitial cells. However, the different spermatogenic cells of the seminiferous tubules and
Sertoli cells are not distinguishable. Scale bar: (A,C-H) 50 lm, (B,I) 100 lm.

© 2017 Anatomical Society

 Removing formaldehyde from veterinary gross anatomy, M. Lombardero et al. 315
Fig. 4 Continued.
Silva et al. (2007) stated that their embalming solution
choice (modified Larssen solution) gave satisfactory results
and enabled the use of embalmed dogs in anatomy classes
as well as for practising most operative procedures. How-
ever, they discussed the fact that the embalming solution
has to be improved to allow better preservation of the
abdominal viscera. Similarly to Janczyk et al. (2011), who
used nitrite pickling salt as an alternative to formaldehyde,
the abdominal organ appearance changed dramatically
unless the abdominal cavities were opened. Taking our
experience and these data together, it seems that the main
problem is not the fixative but the method of perfusion.
Finding a suitable perfusion method will allow a perfect
embalming of both the musculoskeletal system and viscera.
The solution to this inconvenience may be to open a pas-
sage through the body wall to let the brine bath reach the
body cavities, but this has not yet been attempted.
In reference to the SSS method used by Hayashi et al.
(2014), in addition to the hazardous compounds still pre-
sent in the formula (formaldehyde and phenol), the
other disadvantage is that they introduced up to 25 L of
SSS into each cadaver. Thus, it is rather predictable that
© 2017 Anatomical Society
a considerable amount of fluid would accumulate in the
body cavities. Their article is quite confusing because they
only report the use of 1 L of embalming fluid in the leg
and 5 L in the cephalad-placed cannula in the materials
and methods section, instead of the 25 L mentioned in
the discussion section. In contrast, our method consists of
salting the specimen from the inside due to recirculation
of the SSS, instead of introducing a huge amount of SSS
at one time.
Another use of the SSS solution is for storing organs
already fixed in formalin (and previously washed) at room
temperature. No smell nor hazardous effects are generated
because there is no formaldehyde evaporation. After stor-
ing, organs should be washed out before use; thereafter
they can be soaked in the same brine until the next use.
The SSS was also used to display different organs (previously
embalmed, dissected and labelled) in transparent containers
filled with SSS to show them as a collection item at the
anatomical museum (Fig. 1D), which is at the disposal of
undergraduate students throughout the course.
The SSS method had some inconveniences. One was mas-
sive hair loss from the specimen after spending some time
316 Removing formaldehyde from veterinary gross anatomy, M. Lombardero et al.
Fig. 4 Continued.
immersed in the SSS bath. When detected, hair in suspen-
sion is easily collected manually with a sieve. Another is the
corrosion of metal by the salt. Therefore, metal containers
should be avoided. Corrosion of stainless steel surfaces, such
as dissection tables and trolleys, can be avoided by properly
rinsing with tap water after each use. No other corrosive
effects, such as staining stone terrazzo floors, as reported
by Janczyk et al. (2011) for nitrite pickling salt, were pro-
duced by our SSS. When drops of the solution dry, the salt
crystallises, so it is easy to see and be removed effortlessly
when cleaning.
This proposed SSS method allowed us to achieve the fol-
lowing main points:
(1) To keep animal cadavers in good condition until the
end of practical sessions. The long-term advantage
is that it reduced the number of animals for teach-
ing purposes.
(2) To preserve the specimen while maintaining the
appearance, consistency, and flexibility of fresh tis-
sue, also preventing offensive smells.
(3) To avoid the use of and exposure to hazardous com-
pounds and biological fluids by teaching staff and
students.
(4) To reduce spending with the use of common
products.
Conclusions
The use of SSS is a valid method for the preservation of
specimens used in the practical teaching of veterinary
gross anatomy. It is an inexpensive technique and much
healthier for users than traditional formalin. In addition,
it allows the prolonged preservation of specimens, main-
taining a similar appearance and consistency to fresh
material. This would undoubtedly be the ideal method
for veterinary anatomy specimens, as long as a suitable
perfusion method that perfectly embalms both muscu-
loskeletal tissue and viscera is found, which should be
minimally invasive in order to preserve the physical integ-
rity of specimens before use.
Conflict of interest
A poster communication about the same topic entitled
‘Innovation and improvement of Anatomy practical ses-
sions: replacement of formaldehyde by saturated saline
© 2017 Anatomical Society
 Removing formaldehyde from veterinary gross anatomy, M. Lombardero et al. 317
solution’, was presented to the III Congreso de Docencia
 rdoba (Spain) in June 2016.
Veterinaria VetDoc held in Co
Acknowledgements
This collaboration was possible thanks to IACOBUS Program 2014
(1st edition) as part of the activities from the ‘Agrupacio n Europea
de Cooperacio  n Territorial Galicia–Norte de Portugal (GNP-AECT)’.
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