When summarizing the long history of embalming, one has to identify the main purposes for which cadavers were embalmed. One of the first and overall a very important motive was religious beliefs. In several ancient cultures, not only the Egyptian culture, eternal life was associated with a preserved body; those whose body decayed would be excluded from the afterlife. This was supported by the fact that bodies did not decompose when buried under certain circumstances in which natural preservation took place. These natural means of preservation comprise freezing, desiccation or exsiccation, either by dry heat or dry cold, or the specific nature of the soil at the burial site (Johnson et al. 2012). Coastal hunter-gatherers in the Atacama Desert of northern Chile and southern Peru, known as the Chinchorro culture, were among the first to perform artificial mummifications (Marquet et al. 2012). Under a scenario of increasing population size and extreme aridity (with little or no decomposition of corpses), dead individuals may have become a significant part of the landscape, creating the conditions for the manipulation of the dead that led to the emergence of complex mortuary practices as early as 5000–6000 BC (Marquet et al. 2012). Based on the empirical knowledge, the techniques of preservation were enhanced; in Egypt starting as early as in the first dynasty c. 3200 BC. Specialized persons were in charge of these activities; these were – or became therefore – members of the priest caste. Two major developments characterized the transition from the utilization of mere natural means of preservation to sophisticated embalming procedures performed by these priests: first of all the use of additional means such as natron, herbs, cedar oils, natural, tree-derived resins, incense and gums, pitch, and tar, and secondly the introduction of the exenteration or evisceration. This exenteration characterized the preservation of human remains for the next millennia. There are hints that also cadavers buried at the Royal Cemetery of Ur in the late Early Dynastic phase (c. 2500 BC) were preserved by means of heat and mercury (Baadsgaard et al. 2011).
Another method described was immersion in honey, which mainly descended from the Persians, with Alexander the Great being the most prominent cadaver treated in this way. The embalmment of Alexander reveals an additional purpose for body preservation: the necessity for a long-distance and long-term transportation, in Alexander's case, the transfer from Babylon to Alexandria. This technique was re-evaluated in 2004 (Sharquie & Najim, 2004). Whether the Ptolemaic scientists and ‘anatomists’ (first half of the third century BC), Herophilus of Chalcedon and Erasistratus of Ceos, used embalming techniques for their dissected cadavers is not known (Longrigg, 1988).
More or less sophisticated techniques of embalming are known from ancient Ethiopians, the Guanches of the Canary Islands, Peruvians, the Jivaro Indians of the Marano River in Ecuador, the Indians of Central America – Aztecs, Toltecs, and Mayans – and North America, and the inhabitants of the Aleutian Islands and the Kodiak Archipelago (Mayer, 2012), and also Tibetans and Nigerian tribes (Ezugworie et al. 2009). Ancient people of Ogoni, Nigeria, predominantly used large quantities of alcohol concentrate, potash, herbal leaf (Ocimum gratissimum, African basil) and kernel oil (Udoaka et al. 2009).
The hitherto oldest known form of artificial preservation in Europe has been found in the dolmenic burial ‘La Velilla’ in Osorno (Palencia, Spain; Martin-Gil et al. 1995). There, 5000-year-old human bones have been found, which were carefully covered by pulverized cinnabar (vermillion), which ensured their preservation. The authors believe that the vermillion was deliberately deposited for preservative purposes as no cinnabar mine is to be found within a range of 160 km and large amounts (hundreds of kilograms) were used, and as its composition, red mercuric sulphide, is similar to that of preparations used in technical embalming. Nevertheless, embalming remained unusual in Europe, with some reported exceptions during the time of the Roman Empire. The presence of chemical components, such as sesquiterpenes, triterpenoids, and diterpenoids, originating from coniferous and pistacia resins, myrrh, and other spices, found in a partially mummified body dating to AD 300 found in Northern Greece, confirm ancient information on preservation methods of the deceased in Greek and Roman times (Papageorgopoulou et al. 2009).
In China deceased people were obviously embalmed (Brown, 2002), with the main example of Xin Zhui, the Lady of Dai of the Western Han Dynasty, who died between 187 and 145 BC (Chunhong, 2004). Her corpse was found in 1971, when workers were digging an air raid shelter near the city of Changsha. Her remains were extraordinarily well preserved, to pave her way to immortality, but the methods of embalming, and especially the liquid in which Xin Zhui was immersed, are still unknown. To intensify the mystery, two other tombs containing bodies in a similar state of preservation have been found within a few hundred miles of Xin Zhui. One was a magistrate by the name of Sui and the other was Ling Huiping, the wife of a powerful Han Dynasty lord.
Several other well-preserved mummies such as the Iceman from the Similaun glacier (Seidler et al. 1992) or the bog bodies (Glob, 2004; Anonymous, 2012 (embalming essay)) cannot be accounted for as an intended preservation.
Period of anatomists
From those ancient cultures, embalming spread to Europe, where, in time, it became a widespread practice. Descriptions of methods used in Europe for almost 1200 years, starting at about AD 500, have been preserved in the writings of contemporary physicians, such as Peter Forestus (1522–1597) and Ambroise Paré (1510–1590; Table 1). Forestus described his procedure as follows: eviscerate the body, wash with cold water and aqua vita, fill cavities with consecutive layers of Aqua vita moistened cotton, and powder (Table 2), sew the corpse, and finally wrap the corpse in waxed cloth and other things.
Table 1. Paré's components.
|Aqua vita||Radix pul rosar, Chamomile, Balsami, Methe, Anethi, Salvia, Lavendula, Rorismar, Marjoran, Thymi, Absinthi, Cyperi, Calami aromat, Gentiana, Irosflorent, Accavederata, Caryophyll, Nucis moschat, Cinamoni, Styracis calamita, Benjoini, Myrrha, Aloes, Santel|
|Strong vinegar, boiled with|
|Wormewood (Artemisia absinthium)|
Table 2. Embalming powder used by Peter Forestius.
|Prepare a powder from|
|2½ lbs aloes|
|1½ lbs myrrh|
|7 handsfull of ordinary wermut|
|4 handsfull of rosemary|
|1½ lbs pumice|
|4 lbs majoran|
|2 lot storacis calamata (≈ 1/16 lbs, 30 g)|
|½ lot zeltlinalipta muscate|
Embalming during the Middle Ages included evisceration, immersion of the body in alcohol, insertion of preservative herbs into incisions previously made in the fleshy parts of the body, and wrapping the body in tarred or waxed sheets. Later on, in the renaissance period, embalming became influenced by scientific developments in medicine (Ezugworie et al. 2009). Bodies were needed for dissection purposes and preservation required more refined embalming techniques. Among these new techniques, there was the injection into hollow structures of the body, but normally not into the vascular system. Nevertheless, several attempts to inject the vascular system have been passed down; for example, Alessandro Giliani of Persiceto, who died in 1326, used an arterial injection of coloured solutions that later hardened (da Vinci & O'Malley, 1983). Leonardo da Vinci (1452–1519) described a method of preserving the cadavers that he studied. His embalming fluids were mixtures made from turpentine, camphor, oil of lavender, vermilion, wine, rosin, sodium nitrate, and potassium nitrate (McKone, 1999). Da Vinci also used an injection of wax to the ventricles, Jacobus Berengar (1470–1550) injected warm water into veins, Bartholomeo Eustachius (1520–1574) is said to have used injections of warm ink, Reinier de Graaf (1641–1673) injected different liquids and added mercury (de Graaf, 1668), and Jan Swammerdam (1637–1680) injected a wax-like material that later hardened (Mayer, 2012).
Another famous scientist known to embalm by injecting a prepared preservative chemical solution, liquor balsamicum, into the blood vessels was Frederik Ruysch (1638–1731), but his technique was unknown for a long time (Mayer, 2012). In 1717, Ruysch sold his ‘repository of curiosities’ to Peter the Great for 30 000 guilders, including the secret of the liquor, which, according to a recently published book, contained clotted pig's blood, Berlin blue and mercury oxide (Driessen-Van het Reve, 2006). After a first visit to Ruysch, Peter the Great wrote: ‘I saw boys and girls 4 years old, visibly well vascularized, with open eyes and soft little bodies, and they were not even in alcohol.’ (Driessen-Van het Reve, 2006). Another Dutch scientist, Stephen Blanchard (1650–1720), published his embalming method in 1688 (Mayer, 2012).
With the progress made in embalming by arterial injection, research for new preserving fluids opened up another possible way to extend this scientific field of expertise by means of chemistry (Trompette & Lemonnier, 2009). During the 19th century, British, French and Italian scientists perfected such techniques, thereby enabling them to reach every part of the cadaver. Among those British scientists were William Hunter (1718–1783), John Hunter (1728–1793) and Matthew Baillie (1761–1823), who all used an arterial injection of several oils, mainly oil of turpentine, to which they added Venice turpentine, oil of chamomile, and oil of lavender (Table 3). Vermillion was intentionally used a dye, but would have added additional preservative potential to the final solution (Mayer, 2012).
Table 3. Solutions and powder used by W. Hunter.
|Oil of turpentine||Camphorated spirits of wine|
|Added Venice turpentine|
|Oil of chamomile|
|Oil of lavender|
|Portion of vermilion dye|
|Camphor|| ‘Essential’ oils of rosemary and lavender|
In France, several different approaches were developed and used. Cuvier (1769–1832) used pure alcohol, Chaussier (1746–1823) immersed eviscerated bodies in a solution of dichloride of mercury, Thenard (1777–1857) injected an alcoholic solution of dichloride of mercury, and Sucquet (1840–1870) used a 20% zinc chloride solution. Jean Nicolas Gannal (1721–1783) started his career as an apothecary's assistant and became the first to offer embalming to the general French public (Mayer, 2012). His research was not restricted to scientific and medical activities but also covered funeral embalming, using simplified methods that did not involve lacerating the corpse (Trompette & Lemonnier, 2009). In fact, he was the first embalmer to perform documented scientific studies in the field of embalming, which he published – almost completely – himself (Table 4; Gannal, 1840). The final formula was patented and secured, but his successful embalming fluid contained a solution of acetate of alumina (Mayer, 2012).
Table 4. Gannal's experimental arsenal.
|Acids: acetic – arsenous – nitric – hydrochloric|
|Alkali salts of copper – mercury – alum|
|Various combinations: alum, sodium chloride, nitrate of potash, acetate of alumina, chloride of alumina|
In Italy, Guiseppe Tranchina (1797–1837) was a famous anatomist who openly advocated and successfully used arsenic solutions for arterial injection (Mayer, 2012). History has it that his technique was the very first documented method that did not involve evisceration. One of his successors, not as anatomist but as embalmer, was Alfredo Salafia (1869–1933; Piombino-Mascali, 2009). He embalmed several important persons, but his most prominent body was Rosalia Lombardo, an Italian child born in 1918 in Palermo, Sicily. She died of pneumonia on 6 December 1920. She was embalmed and her glass-covered coffin was admitted to the Capuchin catacombs of Palermo in Sicily. For a long time, it was suggested that his fluid might contain arsenic. The recent discovery of a hand-written manuscript by Salafia himself revealed that his solution was one of the very first formulas that included formaldehyde (Table 5; Salafia, c. 1927; Piombino-Mascali et al. 2009).
Table 5. Salafia's solution (Salafia, ca. 1927).
|One part of glycerine|
|One part of a solution of formalin (40%) saturated with zinc-sulphate and 10% of dry zinc-chloride|
|One part of a solution of alcohol saturated of salicylic acid|
One of the last anatomists who openly published a report of an embalming fluid containing arsenic, was Edmond Souchon. His formula A contained 1.5 gallons of water, 1 gallon of arsenious acid (saturated solution) and 8 oz of 40% formaldehyde; this solution was mixed with formula B containing 16 oz of alcohol, 8 oz of carbolic acid (liquefied crystals), 16 oz of glycerine, and 2 oz of creosote (Souchon, 1908).
Modern anatomical preservation
Prior to the introduction of carbolic acid, or phenol, and later of formaldehyde, the main preserving agents used in anatomies were alcoholic solutions of arsenic and/or alumina salts in different concentrations. Most of these ‘modern anatomical embalming fluids’ are summarized in Supporting Information Table S1. Table 6 gives a comparison of different embalming techniques in terms of advantages and disadvantages, long-term storage and usability for anatomical teaching.
Table 6. Comparative table of different techniques.
|Salafia (c. 1927)||Longterm storage||Toxic||Extremely well, when the coffin is sealed||Not tested|
|Kaiserling (Pulvertaft, 1950)||Good preservation of colour and form||Only for isolated specimens||Not applicable||Not applicable|
|Jores (1896, 1913)||Easy storage||No data available||Satisfactory||Satisfactory|
|Woodburne & Lawrence (1952)||Very active as fungicidal agent; soft and plastic; cheap||Medium brown colour||No data available||Highly satisfactory|
|Peters (1956)||Good preservation of intestines; does not affect the dissector's skin; odourless; objects sty smooth and elastic; colour-preserving||No data available||Possible||Satisfactory|
|Erskine (1961)||Soft and flexible, less exsiccation||No data available||Satisfactory||Satisfactory|
|Richins et al. (1963)||Decreased rigidity; increased bactercidity and fungicidity; less browning||No data available||Successful for 2 years||No data available|
|Dayton et al. (1965)||No data available||No data available||No data available||No data available|
|Beck (1966)||No data available||No data available||No data available||No data available|
|Tutsch (1975)||Cheap; odourless||No data available||No data available||Satisfactory|
|Bradbury & Hoshino (1978)||Moderate degrees of movability […] and adequate degree of hardness […] for dissection||No adequate fixation of brains||No data available||Satisfactory|
|Platzer et al. (1978)||Increased fungicidity; cheap||No data available||Almost unlimited, when vacuum packed||No data available|
|Logan (1983)||Soft preservation; obviates excessive noxious fumes||No data available||Satisfactory||Facilitates micro-dissection|
|Frølich et al. (1984)||Soft and flexible||Slight odour, headache, drowsiness; mild eye, nose and throat irritation||Up to 10 years||‘Suitable’|
|Frewein et al. (1987)||Smooth, colour-preserving||Fluid accumulations||No data available||Satisfactory|
|Ikeda et al. (1988)||‘Well fixed’||No data available||No data available||Satisfactory|
|O'Sullivan & Mitchell (1993)||Formaldehyde vapour levels below COSHH limits; improved tissue preservation; more nature coloration||No data available||Proved up to 2.5 years||Satisfactory|
|Macdonald & MacGregor (1997)||Less toxic||Grey hue of skin and muscles||No data available||Satisfactory up to 6 month|
|Coleman & Kogan (1998)||Excellent preservative properties; minimal structural distortion; tissue supple; little desiccation; natural colours||No data available||No data available||Satisfactory|
|Thiel (1992, 2002)||High colour preservation, smooth and flexible||Expensive; Disintegration of muscular tissue; limited time for dissection||No data available||High acceptance|
|Powers (2003)||No data available||No data available||No data available||No data available|
|Silva et al. (2007)|| |
Modified Larssen: good coloration, odourless, in vivo-like flexibility
|Laskowski: dark, loss of tissue texture, skin desquamation, odour||No data available|| |
Laskowski: less suitable for skin or oral cavity surgeries
Modified Larssen: well accepted by students
|Barton et al. (2009)||Smooth||No data available||No data available||High acceptance|
|Mills (2010)||High mould preventiong||No data available||No data available||No data available|
|Al-Hayani et al. (2011)||No structural distortion, not colour changes||Hardening outside the tank; > 2 days for re-softening||When waxed, possible||No data available|
|Anichkov et al. (2011)||Natural appearance, odourless||No data available||Up to 1.5 years||No data available|
|Janczyk et al. (2011a)||Neutral smell||Yellowish coloration; corrosion; Disintegration of abdominal organs||Up to 1 year||Limited usability|
|Hammer et al. (2012)||Flexible tissues, aesthetic appearance; less toxic||Expensive||Up to 3 years||No data available|
|Shi et al. (2012)||Less toxic, good preservative properties, low volatility|| ||Up to 2 years||No data available|
|Goyri-O'Neill et al. (2013)||Good coloration and flexibility||No data available||No data available (good short term preservation ≤ 6 month)||No data available|
Phenol was introduced to anatomical embalming by Laskowski (1886) in the mid-19th century. He initially used a mixture of phenol and glycerine as vehicle (one part phenol, 20 parts glycerine); later on he replaced parts of the glycerine with alcohol (one part phenol, one part boric acid, four parts alcohol, 20 parts glycerine). A similar formulation was developed some years later independently by Rüdinger in Munich (Grönroos, 1898). Alternatively, oxyquinoline (chinosol; 0.63%) was used as single chemical for injection purposes (Schiefferdecker, 1897).
A leap forward came with the discovery of formaldehyde by the German chemist August Wilhelm von Hofmann in 1869 (Hess, 1901). It was determined to be an excellent preservative (Trillat, 1892; Blum, 1893, 1894, 1896; Gerota, 1896) and became the foundation for modern methods of embalming (Ezugworie et al. 2009). Within a few years, until 1898, eight of 45 medical schools throughout Europe introduced formaldehyde for preservation purposes (Grönroos, 1898). Even at that time, there was discussion about the final concentration, with some authors advocating concentrations as low as 3%, others demanding 10%. In addition, the immediate adverse effects were already known: skin irritation, conjunctivitis, irritations of the respiratory system, and headache. Overall, Grönroos summarizes, formaldehyde is not appropriate as a solitary preservation agent.
Up to now, several modified formulae have been published in the scientific literature.
Kaiserling's method for the preservation of the colour and form of specimens, published in 1897, is still widely used (Supporting Information, Table S2); nevertheless, this method is mainly usable for isolated (organ) specimens and is not suitable for anatomical dissection, when the complete method is used (Pulvertaft, 1950). Specimens are fixed in Solution I for up to 2 weeks, depending on their size. Larger specimens should always be injected. In this solution the colour contrasts disappear and are to some extent restored by the ethyl alcohol, wherein the specimens should remain for periods of up to 1 h, but must be carefully watched to ensure that they are removed when the optimum stage is reached; if kept for longer periods, the colour fades (again) and cannot be restored. Solution III is the mounting fluid, which is obsolete for dissection purposes.
Another well-known fixative solution was developed by Jores, containing Karlsbad salts, chloral hydrate and formaldehyde (Supporting Information, Table S3; Jores, 1896, 1913; Bradbury & Hoshino, 1978).
Woodburne & Lawrence (1952) investigated an improved embalming fluid formulation, based on their usual alcohol-glycerine-phenol-formaldehyde embalming formula. Glucarine B (Glyco Products Company Inc., Brooklyn, NY, USA), a commerical sorbitol formulation, was found to be an entirely satisfactory replacement for glycerine. Isopropanol seemed to be the logical substitute for ethanol. Woodburne and Lawrence tested eight different fluids for their germicidal activity against Mycobacterium tuberculosis, Staphylococcus aureus, Eberthella typhosa, Pseudomonas aeruginosa, Proteus vulgaris, Bacillus anthracis, Clostridia tetani and novyi, β-haemolytic Streptococcus pyogenes, and for their fungicidal activity against Penicillium notatum, Aspergillus niger, Coccidioides immitis, Histoplasma capsulatum and Cryptococcus neoformans, with excellent results for the formulation given in Supporting Information, Table S4.
Peters described modifications of the Jores' solution (Peters, 1956). These immersion fluids are generally free of formaldehyde and phenol, which are replaced by choralhydrate (Supporting Information, Table S5); nevertheless, Peters adds 2% phenol for the preservation of pancreas, stomach and intestines.
Erskine described an embalming fluid used in Dublin (Supporting Information, Table S6) which is reported to provide excellent properties of embalmed cadavers for dissection over 3 years (Erskine, 1961). Besides the common shares of ethanol, formaldehyde, glycerine, and phenol, this fluid also contains sodium arsenate, salicylic acid and 6-chlorthymol, the latter to provide appropriate fungicide properties.
Richins et al. (1963) presented an improved embalming fluid, which uses potassium pyrophosphate and magnesium chloride to decrease the rigidity associated with formalin fixation (Supporting Information, Table S7). Furthermore, they substituted phenol with sodium pentachlorophenate, which improved colour relationships and eliminated most of the unpleasant cadaver odour. Finally, sorbitol replaced glycerine as a humectant with less browning of tissues, and a wetting agent was incorporated to facilitate distribution and penetration of the fluid.
Within their study on the influence of diet upon the composition of tissues and atheromata, Dayton et al. (1965) noted an embalming fluid consisting of sodium carbonate monohydrate 16 g, sodium borate 53 g, formaldehyde (37%) 200 mL, diethylene glycol 118 mL, eosin Y 0.16 g, Aquarome Special (unknown commercial product) 5.3 mL, Igepon 1.7 mL (sodium 2-sulphonatoethyl laurate, an anionic surfactant), and water to make 1 L. This fluid was used for embalming whole cadavers prior to pathological dissection.
Beck (1966) stated that the diffusing properties of arterial embalming fluids that contain formaldehyde as a prime preservative can be vastly improved when they also contain relatively small amounts of a substantially neutralized polyacrylic acid (0.005–0.5%). Furthermore, paradichlorobenzene and/or orthodichlorobenzene (0.025–5%) in embalming fluids and solutions should provide an unusual degree of penetration and outstanding preservation.
In 1975, Tutsch (1975) published an embalming fluid formula replaced phenol with Lysoformin® (Lysoform, Berlin, Germany). According to the maufacturer's product sheet, Lysoformin® contains 6.0 g of formaldehyde and 1.8 g of glutaraldehyde per 100 g (Lysoform Dr. Hans Rosemann GmbH, Berlin, Germany); thus, this embalming fluid is completely free of aromatic substances (Supporting Information, Table S8).
In 1978, two different embalming methods were published simultaneously. Bradbury & Hoshino (1978) published their ‘improved embalming procedure for long-lasting preservation of the cadaver for anatomical study’. Prior to the effective embalmment, they treated the cadavers by injecting a blood clot disperser (a diluted commercial product), and then injected 5–6 gal (22.730–27.277 L) of embalming fluid (Supporting Information, Table S9) together with draining of the blood from the internal jugular vein. They did not apply immersion, and the cadavers are stored in a walk-in cold room at 5°C, wrapped in plastic bags.
Platzer et al. (1978) described a preservation system with arterial injection of 3% phenolic acid and 4% formalin in deionised water (110–120 mL kg−1 cadaver weight) and immersion in 2% phenolic acid in deionised water for 1–3 month (Supporting Information, Table S10). Final storage is managed by sealing the fixed cadavers in plastic foils.
In 1983, Logan (1983) described a cadaver preservation procedure which differs in several important features from methods in common use. Fresh cadaver, deep-frozen at −35 °C, thawed for 2 days, then partial flushing of the venous system was effected by infusing a normal saline blood diluent. Arterial infusion and local injection of a preservative solution followed. His solution comprised alcohol, glycerine, phenol, and low formaldehyde, but no quantities were given.
Coleman & Kogan (1998) used almost the same chemicals (they replaced alcohol by isopropyl alcohol), but added a vast amount of sodium chloride (Supporting Information, Table S11). They argued that the high salt content retained in the tissues prevented any further significant desiccation. Salts have also been used in Basel (Supporting Information, Table S12; 4% of sodium choride, and 1% of anhydrous calcium chloride; Kurz, 1977/1978), and Bergen (Supporting Information, Table S13; 5% of potassium nitrate; Frølich et al. 1984).
In Zurich, Frewein et al. (1987) experimented with modifications of the basic recipe by Kurz. Their final modification contains formaldehyde, choral hydrate, calcium chloride, and Almudor® (ISS pest Control AG, Dietikon, Switzerland; apparently discontinued), a disinfecting mixture of formaldehyde, glyoxal and glutaraldehyde (Supporting Information, Table S14; Saupe et al. 2007).
Another embalming fluid, presented in a study of arterial patterns in the hand, consisted of 95% ethyl alcohol (7.6 L), 35% formalin (1.3 L) as a fixative, diethylene glycol (2.7 L) as a preservative, liquefied phenol (1.3 L) as a mould preventative, and water (8.0 L; Ikeda et al. 1988). It seems that this embalming fluid is, or at least was at that time, the common formulation used at Kawasaki Medical School in Kurashiki City, Okayama, Japan.
Thiel (1992, 2002) presented a delicate method for ‘the preservation of the whole corpse with natural colors’. This method has, as stated by the author, the advantage of meeting high standards of preservation without releasing harmful substances into the environment. Nevertheless, his method is quite complicated and includes several problematic and expensive substances during the process of preservation itself. In addition to the basic solutions, the infusion/visceral solution, and the storage solution (Supporting Information, Table S15), Thiel suggests injecting a mixture of 40 mL tap water, 45 mL ethanol and 15 mL formaldehyde to the ventricles of the brain.
To reduce the final formaldehyde concentration, phenoxyethanol can be used to wash out excessive formaldehyde from cadavers (Owen & Steedman, 1956, 1958; Spence, 1967; Frølich et al. 1984; Wineski & English, 1989). Nevertheless, there is no report using phenoxyethanol as primary agent in arterial injection solutions, but there are two US patents by Campbell & Margrave (1995, 1998). According to Campbell and Margrave, a preferred formulation should include glutaraldehyde from about 0.5% to about 2%, an aromatic ether of ethanol (e.g. phenoxyethanol) from about 1% to about 3%, a humectant (e.g. a polyhydric alcohol, 1,2-propanediol or hexylene glycol) from about 5% to about 9%, and an alcohol (e.g. ethanol) from about 27% to about 37% (Campbell & Margrave, 1995). In addition, a buffer and/or anti-oxidant may be included to maintain the stability of the glutaraldehyde. The buffer would adjust the pH in the range of pH 7–9. In addition, a biocide such as benzalkonium chloride or other quaternary ammonium compounds may be added further to deter microbial growth.
O'Sullivan & Mitchell (1993) examined the composition of the embalming fluids from 16 medical schools in the United Kingdom and found wide variation in the proportions, but not the identity, of the constituents of the embalming fluids. All of these medical schools in fact used formaldehyde, industrial methylated spirits1 water, phenol and glycerol, with the proportion of phenol appearing to be a constant feature in all formulae, reflecting its important disinfectant quality. In advance, the authors experimented with several concentrations of the same basic substances, either buffered with 0.075 m phosphate buffer (pH 7.4) or unbuffered (O'Sullivan & Mitchell, 1993). They found that the buffered solutions were ineffective because the pH changed from that of the original buffer to the pH of the embalming fluid itself. The concentrations of their suggested ‘new’ Southampton embalming fluid are given in Supporting Information, Table S16. Formaldehyde vapour level determination in their experimental fluid composition embalming was in all instances within the limits set by the ‘Control of Substances Hazardous to Health’ (COSHH) regulations.
To adopt the embalming fluids for purposes for plastination, Pretorius increased the contents of ethanol (28 L), formaldehyde (1.2 L) and glycerine (0.8 L), and reduced the phenol content (1.2 L); this mixture is diluted with 8 L of water (Pretorius, 1996).
The stock solution used at the Robert Wood Johnson Medical School (Piscataway, NJ, USA) contains three parts propylene glycol, three parts ethanol (95%), and one part phenol (90%) (Macdonald & MacGregor, 1997). The final embalming solution is prepared by adding 810 g potassium nitrate, 567 g sodium borate, and 3.8 g sodium lauryl sulphate to 25 L of hot tap water. Finally, 12.5 L of the stock solution is added to the dissolved salts. Sodium lauryl sulphate is used as a surfactant and should enable the embalming fluid to enter all areas of the cadaver.
At McMaster's University at Ontario (Canada), a complex set of solutions is used (Supporting Information, Table S17; Powers, 2003).
In 2007, Silva et al. compared a modified Laskowski-solution with a modified Larssen solution. The modified Laskowski solution was composed of 800 mL glycerine, 200 mL ethanol, 50 g ‘phenic acid’ (phenol) and 50 mg boric acid (Rodrigues, 1998; Silva et al. 2007). Their modified Larssen solution included 100 mL of 10% formalin, 400 mL glycerine, 200 g chloral hydrate, 200 g sodium sulphate, 200 g sodium bicarbonate, 180 g sodium chloride and (in the final working solution) 9.5 L of distilled water (Guimarães Da Silva et al. 2004). The original solution formula of Larssen from the Hospital Cochim, Paris, was reported by Sampaio to be composed of 500 g sodium chloride, 900 g sodium bicarbonate, 1000 g chloral hydrate, 1100 g sodium sulphate and 500 mL of a solution of 10% formalin and 1 L distilled water (Sampaio, 1989). Sampaio used one part of this solution with five parts of distilled water. We could find no further evidence of this Larssen solution.
In the same year, Constantinescu et al. (2007) noted another formulation of well known ingredients: 1200 mL formaldehyde, 400 mL propylene- or ethylene-glycol, 1000 mL phenol, and water added to 20 L.
Barton et al. (2009) described a ‘soft-preservation fluid’ containing 2 L of phenol (80% aqueous solution), 8 L of industrial methylated spirits,1 8 L water, and 4 L glycerol, for arterial injection.
Investigating a fixation–preservation salt solution containing 23% of nitrite pickling salt, 30% ethanol and 20% Pluriol® E 400 (a mixture of polyethylene glycols), Janczyk et al. (2011a) found it suitable for the preservation of (animal) cadavers with opened abdominal cavity, but not for cadavers, which had a closed abdominal cavity. In these cadavers, the abdominal organs changed their consistency and colour dramatically.
The Anatomy Department of the University of Sydney, Australia, reported in 2010 that two distinct formulations of embalming fluids were being used (Supporting Information, Table S18; Mills, 2010). Formula (A) is routinely used for preserving cadavers destined for the dissecting room or prosected specimens. The combination of pine oil, phenol and particularly di-(2-hydroxyethoxy)-methane in formula (A) has almost completely eradicated the problem of mould growth, particularly Penicillium simplicissimum and Penicillium waksmanii. Vigilance is only required for areas of poor fixation, such as gangrenous extremities. Formula (B) is a modified Kaiserling solution and is used for embalming cadavers destined for cross-section and plastination. In both of these applications, the initial high formaldehyde concentration is removed from the finished product. The absence of alcohol makes it easier to freeze the specimen prior to sectioning. With both formulae, at least 20 L of embalming fluid is injected into each body. After injection, the cadaver is washed down with tap water and then sprayed with surface disinfectant (70% alcohol, 5% Dettol, 25% water) and placed in a cold room at 4–6 °C for 12 months prior to use. For moistening purposes, the cadavers or specimens are sprayed intermittently via soaker hoses installed on the walls and roofs of the cabinets with a preservative fluid comprising 1% di-(2-hydroxyethoxy) methane, 1% 2-phenoxyethanol, 30% methylated spirit and 65% water.
Recently, Hammer et al. (2012) described a formaldehyde-free system which comprises ethanol (0.7 L kg−1 body weight), glycerine (5%) and thymol. The ethanol–glycerine fluid is injected arterially; afterwards the bodies are immersed in ethanol (65%). A thymol-ethanol solution (thymol 30.044 g L−1; 10% ethanol in aqueous solution) as moistening solution is used for keeping the state of fixation at room temperature.
Polyhexamethyleneguanidine hydrochloride was used as embalming agent and was compared with the efficiency of formalin fixation by Anichkov et al. (2010, 2011). They used this fixation method to obtain anatomical and histological preparations from human organs and chick embryos at 12 days of development. The anatomical preparations had external appearances similar to those of freshly prepared organs; nevertheless, only organs – not whole bodies – were embalmed.
Another replacement for formaldehyde has been suggested by Shi et al. (2012). Their preservative is a blend of acid, buffer solution and cross-linking agent, Tetrakis(hydroxymethyl)phosphonium chloride, which acts as fungicide, stabilizer and fixative, respectively.
Recently, Al-Hayani et al. (2011; Bedir, 2009), suggested the use of shellac, a complex mixture of aliphatic and alicyclic hydroxyl acids and their polyesters, derived from the hardened secretion of the lac insect(s). The use of shellac had previously been proposed by Pate (1938) for preserving anatomical specimens for museum and teaching purposes. Shellac is soluble in alcohol and alkaline solutions but insoluble in water. It is widely used in the food industry, and in the pharmaceutical industry as an enteric coating material. For their purposes, the authors solved the dry shellac (80 kg) in diluted ethanol (200 L; 58%). Defrosted cadavers were immersed in this solution at a pressure of 15 kPa for 3 days. The authors found that the cadavers could be used in the open air for a long time; however, if kept out of the tank for a period of more than 1 week, they may harden due purely to the hardening of resin. Long-term storage of the cadavers was achieved by spraying the cadaver with a waxed solution. This led to hardening within 2 days. Such cadavers could be stored easily in room conditions. For dissection/examination, re-softening was done by replacing the cadaver inside the softening tank for a couple of days. The gross anatomy of tissues and organs showed neither structural distortion nor colour changes, with tissues remaining supple and easy to dissect; only the skin exhibited brownish glistening discoloration with no colour changes in the subcutaneous structures, even over an extended period.
Goyri-O'Neill et al. (2013) have recently reported an embalming fluid containing nine parts diethylene glycol and one part monoethylene glycol. They used this clear liquid, which they described as practically odourless and colourless, to inject on average 7 L arterially, using a pulsed infusion at a rate of 60–70 pulses per minute. The pressures used are not given but should have been quite high, as the whole injection was performed within 30–45 min. Whereas the authors provide appropriate information on the pre-treatment (external washing with chorhexidine soap and cooling during transport to 4–6 °C), there is no information on the conditions in which the cadavers were stored after injection of the embalming fluid. Macroscopically, this embalming fluid results in good short-term preservation quality (up to 6 months). A histological evaluation 1 month after injection revealed the best results for a striated thigh muscle; the skin was also well preserved, whereas the quality of preservation of the buccal mucosa was not as good.
There are also several proprietary mixes, such as the Dodge and Genelyn solutions, whose exact composition is not available. However, it is known that the Dodge and Genelyn solutions both contain methanol and formaldehyde and dyes but no phenol (Jaung et al. 2011). The AnubiFiX™ method was recently introduced in the Netherlands (Kleinrensink, 2011). This embalming technique is based on a new prerinsing method combined with a normal 4% formaldehyde fixation solution. In contrast to conventional embalming methods, AnubiFiX™ embalming should result in a very small decrease in flexibility and plasticity (Slieker et al. 2012).