As in most European countries, modern natural history studies and collections in Portugal, started in the mid of the18^th century (Brigola 2003; Ceríaco 2021). In the first half of the century, the Portuguese crown and private individuals held natural history specimens in their collections and private cabinets; however, most of these were lost in the tragic earthquake that destroyed Lisbon in 1755 (Brigola 2003). Despite the existence of these collections, the “birth” of Portuguese natural history collections can be directly linked to the arrival of the Paduan naturalist Domenico Vandelli (1735–1816) in the country in the mid-1760s. Vandelli would be hired by the Portuguese crown to be the director of the newly-established scientific institutions – the Royal Botanical Garden and the Royal Cabinet of Natural History. Founded in 1768 in the vicinity of the palace grounds in Ajuda, Lisbon, both these scientific establishments became known as either the Botanical Garden of Ajuda, the Cabinet of Ajuda or simply the Ajuda complex (Ceríaco 2021). The Cabinet of Ajuda was a “modern” natural history museum, with two visitable collections rooms organised following Linnean taxonomy, an associated laboratory, library, warehouse and the “Casa do Risco”, a scientific illustration studio. The collections covered all the traditional realms of natural history – geology, botany, zoology, but also ethnography and anthropology – and the museum rapidly became one the largest natural history collections in Europe. In 1772, Vandelli would also become the first professor of Natural History in the reformed Universidade de Coimbra, establishing a natural history museum in the university to support his classes. While holding the two most important positions in natural history in the country, Vandelli would still have the time to participate in the creation of the Royal Academy of Sciences of Lisbon and actively participate in the national political scene.

To enrich the collections of the institutions under his care, Vandelli wrote several naturalist instructions to both his students and to the members of the learned societies. His first instructions, an internal unpublished manuscript, were entitled “Viagens filosóficas ou Dissertação sobre as importantes regras que o filósofo naturalista, nas suas peregrinações deve principalmente observar” [translation: Philosophical voyages or Dissertation about the important rules that the naturalist philosopher, within his trips, shall observe] and presented some of the first examples of fluid preservation reported in the country (Vandelli 1779). According to Vandelli (1779) for small mammals such as mice, the specimen would be cut open near the anus to remove all the viscera, which would be replaced by a portion of camphor the size of a pea and then immersed in a jar of spirit where burnt alum had been dissolved. The spirit should not be pure, but composed of two parts of water and one of spirit. For birds, Vandelli suggests that the viscera should also be removed and replaced by camphor, the specimen wrapped in cloth and immersed in wine spirit, corrosive sublimate (“solução de solimão”; also known as mercuric chloride, HgCl₂) and camphor. Reptiles and amphibians should simply be immersed in wine spirit, while fishes should require special precautions as they “corrupt faster” and, therefore, the wine spirit should be stronger and with as much “dissolved potassium alum” as possible. The viscera, especially the liver, should be removed first, as this would “weaken the spirits”. Vermin should be sent in wine spirits.

In 1781, both the Royal Academy of Science of Lisbon and the Royal Cabinet of Ajuda issued naturalist instructions to be delivered to their naturalists and correspondents, in the hope of enriching the collections of both institutions. There is an obvious similarity between both the Academy and Ajuda instructions, as they were most likely produced by the same group of individuals, such as Vandelli or his student Alexandre Rodrigues Ferreira (1756–1815), who would become famous for his philosophical journey in Brazil. Those of Ajuda, entitled “Methodo de Recolher, Preparar, Remetter, e Conservar os Productos Naturais” [Translation: Methods of collecting, preparing, shipping and preserving natural products] were never published, but were certainly well circulated at least within the naturalists of the Ajuda complex. The surviving manuscript bears the handwriting of both Ferreira and another unidentified individual and presents plates illustrating some of the techniques (Anonymous 1781a; Fig. 2). It is believed that this was an internal document used to train the Ajuda naturalists (Ceríaco 2021) and, thus, represents a very valuable source of information regarding the Royal Cabinet’s internal practices. Fluid preservation is mentioned in several passages as a preparation and preservation method for different types of animals. For small mammals, such as “small mice and marmosets”, it was suggested that the specimen would be cut open near the anus to remove all the viscera, which would be substituted by a portion of camphor the size of a pea and then immersed in a jar in spirits. The author notes that it does not matter if it is “wine spirit”, “sugarcane spirit [agoa ardente de canna]” or “cachaça”, but also notes that none of these spirits should be pure, but rather a dilution containing “two parts of water and one part of spirit” where burnt potassium alum has been dissolved. The specimen would then remain for 10 to 12 days in this original solution, which would then be discarded and refilled prior to shipment to the museum. The author also refers that the same method is appliable to fishes, “amphibians” [reptiles included – the class “Reptilia” had only been recently proposed by J. N. Laurenti in 1768] and birds, which in the latter case would need to be wrapped in cloth, leaving only the beak and the tip of the tail protruding from the fluid (Fig. 3). Crabs, lobsters and shrimp should be discarnate and then bathed in strong spirits. All remaining “vermin” would simply be preserved in “wine spirit”.

The instructions issued by the Academy (Anonymous [most likely Vandelli] 1781b) are very similar to those of Ajuda, but, nonetheless, present some small differences. For small mammals, the author also suggests that the intestines should be removed through a small incision near the anus. The insides of the specimen should be then cleaned and dried before being filled with oakum or cotton mixed with “anti-putrefaction drugs” and the eyes should also be removed. After this, the specimen could be preserved in jars with “three parts of pure water and one part of wine spirit, where a good portion of burnt potassium alum has been dissolved”. The same method was suggested for small birds, with the author suggesting crushing the breastbone to avoid deformities and to wrap them in cloth. Still, according to the author, “if the distances are not that big, regardless of the kind of bird, it would be enough that after being prepared by the previous method, they can be submerged for 15 days in strong wine spirit, where a good portion of potassium alum and camphor have been dissolved”. Small herpetological specimens and fishes, would simply be immersed in spirits, where a good quantity of potassium alum and camphor had been dissolved, noting, however, that these spirits should be changed and refilled at least twice before the shipment. Small crustaceans should be carefully wrapped in cloth to avoid breaking their legs and antennae, tied and immersed in a jar with wine spirit. Invertebrates other than butterflies or insects should be preserved in wine spirits similarly to small vertebrates. Interestingly, the author also refers to how the jars should be closed using close-tight lids and sealed with a mix of wax and resin.

In 1783, Agostinho José Martins Vidigal (dates of birth and death unknown), a student of Vandelli at the University of Coimbra, wrote the “Methodo de fazer observaçoens, e Exames necessários para o aumento da Historia Natural, com os meios de preparar, conservar, e dispor nos Museos os diversos productos da Natureza” [translation: Method of making observations and appropriate examinations to foster Natural History, with the methods to prepare, preserve and display the different natural products in museums] (Vidigal 1783). This manuscript is quite interesting, as Vidigal (1783) provides in its introduction a review of the then-available literature on methods of natural history specimens preservation. Vidigal refers the works of several other 18^th century naturalists and scientists, such as Henri-Louis Duhamel du Monceau (1700–1782) “Memoir on the transport of trees, plants and other natural history specimens by sea” (probably referring to Duhamel (1767)), those of the French naturalist M. Marvye (birth and death dates unknown, Marvye (1763)) and the French entomologist René Antoine Ferchault de Réaumur (1683–1757), David Hultman (1732–1798), Linnaeus student who published the thesis “Instructio musei rerum naturalium, quam, consensu experient. et nobiliss” (Hultman 1753), Henrique Andre Nordblad [Erik Andreas Nordblad (1739–1810)], Linnaeus student whose thesis was the famous and highly influential “Instructio peregrinatoris” (Nordblad 1759), the work he referred to as “o Viajante Naturalista, ou instrucções sobre os meios de recolher, e conservar os diversos objectos da Historia Natural” [translation: the wandering naturalist, or instructions on the methods of collecting and preserving different natural history objects] by the English physician John Coackley Lettsom (1744–1815) [most likely “The naturalist’s and traveller’s companion, containing instructions for collecting and preserving objects of natural history and for promoting inquiries after human knowledge in general”, Lettsom (1774)], the works of Casimiro Gomes Ortega (1741–1818), the then director of the Royal Botanical Garden of Madrid, Spain and, of course, those of Vandelli (1779) and the Lisbon Academy of Sciences (Anonymous 1781b).

Regarding fluid preservation, Vidigal provides some interesting and rather unique suggestions. According to the author, “Several are the spirits that can be used to preserve in an incorrupt manner the animals that are placed inside the jars: among these, some are preferred as they do not evaporate too much, others because they do not bleach the colour of the deposited bodies, an effect that some note to happen with wine spirit, which should always be, according to the more exact observations, of vivid colour. The difficulties that require the most attention is how to keep the jars closed in a way that the spirit does not evaporate; waxed papers, bladders, tin and lead foil, plaster, should be used to prevent evaporation. So far, the most efficient method is the following: the jars are built with a “hook” in the bottom to which the specimen is tied; the jar is then filled with wine spirit and closed with an appropriate stopper; after that turn the jar upside down so the deposited specimen stays in its natural position, this way the evaporation is not so common, especially if a good quantity of Hidrargirum [Hydragyrym; mercury] has been put inside the jar, so when its turned upside down, the jar’s mouth becomes completely covered with metal”. Vidigal goes into detail and dissertates about the overall appearance of the jars: “The jar should be of very transparent glass, cylindrical, should have two borders, an inferior one to serve as a base and a top one that can be used to close them. Its size should be smaller or larger depending on the proportions of the preserved bodies. Finally, it should be taken into account that its overall aspect serves as an adornment to the museum”. Regarding the fluid preservation of animals, Vidigal noted that “The small quadrupeds such as rats, as well as the abortions of the larger ones, are preserved in jars full of wine spirit”, while for birds, besides other traditional taxidermic techniques, Vidigal notes that they are “easily conserved in wine spirit”. Continuing to fishes, these “should be deposited in jars full of wine spirit and, thus, will be always useful to select the smaller ones, if they do not differ from the larger ones besides their proportions. It is, however, necessary that they are still fresh at the moment they are deposited, being certain that if they are deposited still alive in the jar, they preserve better the vivacity of their colours”. The “vermin, as the amphibians”, Vidigal writes “can be preserved in wine spirit”.

In the same year, José António de Sá (1756–1819), a judge and fellow of the Royal Academy of Sciences published the “Compendio de observações, que formam o plano da viagem política e filosófica que se deve fazer dentro da pátria” [translation: Compendium of observations that constitute the plan for a political and philosophical voyage to be carried within the fatherland] (Sá 1783). This text, in the spirit of the Linnean “Instructio peregrinatoris”, provided a method to study, record and collect the natural products of the Portuguese territory. Regarding preservation in fluids, the author suggested techniques that were similar to those of the Academy of Sciences and the Ajuda complex. For small mammals, the animal should be gutted through an incision near the anus and the open cavity should be first cleaned and dried and then stuffed with vegetal materials. The eyes should also be removed. Then the specimen should be immersed in wine spirit that should be changed one or two times. For preservation, the specimen should then be placed inside a jar with modified wine spirit, with “three parts of water” and a lot of burnt alum, otherwise “pure would alter and destroy the animal substances”. For small birds, Sá suggested that first “the insects should be removed and camphor introduced” and then the specimen should be immersed in wine spirit with a similar percentage of that used for small mammals, but instead of alum, corrosive sublimate (“solimão”) and camphor should be added. The specimens should also be wrapped in linen cloths and the chest bone broken to avoid any “deformity”. Interestingly, Sá did not provide any fluid preservation method for snakes and other reptiles, which he suggests being skinned and stuffed. Amphibians, however, “due to their small size, cannot be skinned, are preserved in wine spirit similarly to what has been said for mammals and birds”. The same was suggested for small fishes, “with the single difference that the spirit should be stronger and changed more often”. Any insect with “soft tissues” should also be preserved in wine spirit, while for other “vermins”, such as polyps, the method was slightly more complex. Although still preserved in wine spirit, “to extract them from their little houses, the following must be done […] have pure ocean salt water in a jar, where as soon as the hard substrate is taken from the sea can be deposited there. With a lens observe when some parts of these animals leave their “rooms”, which usually occurs after an hour”; as soon as this happens, with the help of some instruments or even with the own fingers, one will quickly extract the animal and put them in wine spirit, to avoid that it disfigures itself before it dies”. A curious case, not entirely related to natural history per se, was that of the “bi-cephalic monster” born in Évora, southern Portugal, in 1788. The said “monster” was a case of conjoined female twin human babies who died shortly after birth and were dissected and “preserved in the spirit of wine” by a local doctor and eventually dispatched to the Ajuda cabinet (Ceríaco 2013).

By the end of the 18^th century, according to a 1794 manuscript catalogue of the collections, the fluid preserved collections held in the Cabinet of Ajuda surpassed the 1100 specimens and were distributed in a few hundred jars (Ferreira 1794). Of these, 12 were human foetuses, five “monstrous pigs”, 438 birds, more than 165 amphibians and lizards, several juvenile turtles and eggs, 155 snakes, more than 320 fishes and eight marine invertebrates. A considerable percentage were originally from the Philosophical Voyage of Alexandre Rodrigues Ferreira to Brazil (1783–1792), but there were also specimens collected by other Ajuda naturalists and/or donated by private individuals. No specific data exists regarding such collections in the Natural History Museum of the Universidade de Coimbra or in the Royal Academy of Sciences, although they certainly existed in the collections (Ceríaco 2021). Very few specimens of this period survive in collections until today. The few examples that can be unambiguously attributed to this period are several “monsters” and some Brazilian reptiles. One of the “monsters” is the above cited Évora “bi-cephalic monster”, whose specimen still survives in the collections of the Museu da Medicina of the Santa Maria Hospital in Lisbon. Its dispersal history through institutions was discussed by Ceríaco (2013). When the specimen was located, it was still partly immersed in a tawny wine solution, presumably its original preservative. The other “monsters” are a seven-legged dog embryo, a two-faced cat and two conjoined human twins described and illustrated by Vandelli’s “Dissertatio de monstris” (Vandelli 1776), a brief essay on the nature of “monsters”. These specimens were located and identified by Ceríaco et al. (2013) in MCUC’s collections and the Museu de Anatomia Patológica of the Universidade de Coimbra (Fig. 4). More recently, Ceríaco et al. (submitted) identified eight specimens of reptiles (mostly snakes) collected by Alexandre Rodrigues Ferreira during his Philosophical Voyage to Brazil, in the collections of MCUC, as well as a specimen of the iguanid lizard Plica plica (Linnaeus, 1758) collected in Brazil by Alexandre Rodrigues Ferreira and ransacked to the Muséum national d’Histoire naturelle of Paris in 1808. This specimen was used as the holotype of Hypsibatus punctatus Duméril & Bibron, 1837 (see Ceríaco 2021). MUHNAC and MHNC-UP do not have specimens from this period and it is unlikely that other smaller collections have any.

The first decades of the 19^th century were considerably traumatising for Portuguese society. In 1807, the Napoleonic armies invaded Portugal, causing the Portuguese royal family to relocate the court to Rio de Janeiro in Brazil, then the most important Portuguese colony. While the French armies were eventually defeated by a Portuguese and English coalition in the summer of 1808, the Royal Family remained in Rio de Janeiro for more than a decade. During this escape, the Portuguese court took with itself libraries, scientific instruments and other cultural heritage to avoid its pillage by the French. While the natural history collections of the Royal Cabinet of Ajuda never left the country, some of the instruments of the Royal Cabinet of Physics travelled with the court (Ceríaco 2021). During the invasion, the collections in Ajuda were visited by the French naturalist Étienne Geoffroy Saint Hilaire (1772–1844) who took some specimens to Paris, where they remain today (Daget and Saldanha 1989; Ceríaco 2021). During these difficult times, little was achieved in Portuguese Natural History other than basic maintenance of collections. With the Portuguese court established in Rio de Janeiro, the Brazilian capital became the de facto capital of the Portuguese Empire. This resulted in the establishment of new institutions in the city, such as a botanical garden and a natural history museum, both created in 1818.

Regarding naturalist instructions, in the first year of the 19^th century, the Portuguese-Brazilian friar and amateur naturalist José Mariano da Conceição Velloso (1742–1811), at the time responsible for one of the most important typographies in Lisbon, the Arco do Cego typography, published the book “Naturalista instruido nos diversos methodos antigos, e modernos, de ajuntar, preparar, e conserver as producções dos tres reinos da natureza” [translation: Trained naturalist in the various methods, ancient and modern, of gathering, preparing and preserving the natural products of the three Kingdoms of Nature] (Velloso 1800), which in great part was composed of a translation of Abbé Manesse’s (death of birth and death unknown) “Traité sur la manière d’empailler et de conserver les animaus, les pelleteries et les laines” (Manesse 1787). While mostly focused on taxidermy, Manesse (1787) and Velloso (1800) also presented some methods of fluid preservation. The text starts to enumerate the challenges of fluid preservation at the time and presentstheir own experiences on the topic, namely solutions containing alum, ammonia salt, nitre and sea salt. For the first, the author noted that if the solution was too strong, the specimen would shrink and become paler, while if it was too weak, the specimen would rot. For the nitro solution, the colours would be “revivified” and the “meat would be better conserved”, especially if the nitro was not too pure and mixed with some parts of sea salt. The ammonia salt would darken and stiffen the specimens, while sea salt would prevent rotting, but the specimen would shrink, stiffen and change colours if kept in this mixture for too long. Manesse would also note that he had tried a solution combining both nitre, salt and alum in more or less equal parts in water, in which he immersed a “human foetus, that was perfectly preserved for more than a year, even if the glass stopper was just a piece of cork”. The experience was carried out with other animals and worked well. According to the author, this solution had the advantage of being cheaper and better preserving the specimens than the wine spirits, but had the inconvenience of freezing during cold weather, which would cause the jars to crack. To avoid this problem, Manesse suggested adding to the solution an “1/8 part of wine spirit”, but that could sometimes lead to a whitening suspension in the solution, which he attributed to the presence of the alum.

In 1819, a set of instructions were printed in Rio de Janeiro, under the direct patronage of the Portuguese Crown and the Ministry and Secretary of State of the Kingdom. The long title of the work is self-explaining of its content and importance: “Instrucção para os viajantes e empregados nas colonias sôbre a maneira de colher, conserver, e remetter os objectos de historia natural. Arranjada pela administração do R. Museu de Historia Natural de Paris. Traduzida por ordem de Sua Magestade Fidelissima, expedida pelo excellentissimo Ministro e Secretario de Estado dos Negocios do Reino do original francez impresso em 1818. Augmentada, em notas, de muitas instruções aos correspondentes da Academia R. das Sciencias de Lisboa, impressas em 1781; e precedida de algumas reflexões sobre a história natural do Brazil, e estabelecimento do Museu e Jardim Botânico em a côrte do Rio de Janeiro” [translation: Instruction to the travellers and officials in the colonies on the method of collecting, preseving and shipping natural history objects. Organised by the administration of the Royal Museum of Natural History of Paris. Translated by order of His Loyal Majesty, directed by the excellent Ministry and Secretary of State of the Kingdom affairs from the original published in 1818 in French. Augmented with notes of the several instructions to the correspondents of the Royal Academy of Sciences of Lisbon printed in 1781; and preceded of several reflections on the natural history of Brazil and the establishment of a museum and botanical garden in the Court of Rio de Janeiro] (Anonymous 1819). While the first 56 pages of the book were dedicated to discussing the overall challenges and plans for the study of Brazilian natural history and the establishment of the Rio de Janeiro museum and botanical garden, from page 57 onwards, it presented a Portuguese translation of the 1818 French booklet “Instructions pour les voyageurs et pour les employés dans les colonies, sur la manière de recueillir, de conserver et d’envoyer les objets d’histoire naturelle” (Anonymous 1818), populated with endnotes with comments and notes extracted from the 1781 instructions. As the authors noted “the perfect conservation of animals in spirit depends on the quality of the latter, on how the specimens is placed inside the jar and on how to close the jars”. For mammals, the authors noted that the smaller specimens could be immersed in wine spirit in glass jars or barrels, while an endnote added that the intestines should be previously removed through an incision made near the anus. The instructions also mentioned that, while birds should be skinned, whenever there were many available specimens, it would also be important to send some immersed in wine spirit. Small fishes and reptiles should also be preserved in wine spirit, as well as insect larvae. Insects, except for butterflies, would also be better preserved in wine spirit as “this way the internal organs are conserved, which will allow its examination whenever possible”. More details were provided for crustaceans and molluscs. The first, especially the smaller ones, should be preserved in wine spirit, but it was crucial to wash them first in freshwater to clean them of any remains of salt, as “without this, the most part of them will spoil”, noting that this was what happened to “many [specimens] of the rich collection made by Péron”. Molluscs should also be sent in wine spirit, but for those which have shells, this should be removed after the animal was euthanised by immersing it in wine spirit. The shell and animal body should then be numbered with the same number and sent along together. Other “molluscs”, namely corals and other type of marine invertebrates should be first washed in freshwater and immersed in wine spirit, with the attention to take notes of the original colours as “in wine spirit, the color of gelatinous animals does not always remain”. Vermin found in the bodies of other animals should also be preserved in wine spirit.

Further in the text, the authors would provide more details regarding the concentration of the wine spirit. According to the authors, their suggestions were based on “a Memory by Mr. Péron, in the 2° volume of the Voyage to Austral Lands”, as “it is known that this naturalist, to whom the King’s Cabinet owns the richest collection of invertebrate animals, managed to perfectly conserve them”. According to the instructions, the wine spirit should be “between 16 to 22 degrees of Beaumé areometer”, stronger than that would “completely destroy the animal colours”. The 22 degrees’ concentration should only be used for mammals and, overall, all liquors would be good, with a special preference to those that were less “ruddy”. The authors noted that before submerging the specimens in the fluid, they should be thoroughly cleaned and washed of all the “mucous and dirt” with a soft brush and the specimen should be hung in the jar not touching the bottom, as if it did, it would become damaged and “corrupted”. Following Perón’s suggestion, the authors refer that animals could be attached by string to a piece of cork which would make them float in the jar. This way “many animals could be placed in a single jar”. If this process is too difficult, the authors suggest that the specimens could be placed inside a net attached to the lid of the jar. On an important note, that was not previously mentioned in the text, the authors mention that “a small incision should be done in the abdomen of vertebrate animal, so the liquor penetrates in the body”. Referring to Péron on the use of camphored wine-spirit, the authors disregard its use as “camphor is very expensive, its dissolution thickens the specimens and makes it more difficult to dissect them”. The fluid should be renewed after a few days to properly fix the specimen.

More details were provided regarding how to ship, identify and accommodate the specimens. For numbering the specimens preserved in fluid, the authors suggested to tie two briefly separated series of knots on a string, where the first series would correspond to the tens, while the second series to the units. For shipping, the authors preferred glass jars to wooden barrels, as in the latter, “regardless the precautions taken, there is always some liquor that leaks through its pores [of the barrel]”. Square glass jars would also be preferable as “they fit better in boxes”. To seal the jars, the authors noted that “cork stoppers are preferable to all other: the glass stoppers break due to the evaporation of the spirit”. These should be then covered in what the authors and Péron named “Lithocolla”, a paste made from:

Ordinary resin

Well powdered red ochre

Yellow wax

Turpentine oil

which should be prepared the following way:

“Use more or less resin and iron oxide, or turpentine oil and wax, depending on if you want the seal to be more or less fragile. In a single text, you will determine the convenient proportions. Melt the wax and the resin. Carefully add the ochre, mixing it fiercely with a spatula. After boiling the mix for 7 or 8 minutes, add the turpentine oil, mix and let it continue boiling. Be careful to prevent these substances from inflaming; in case this happens, have at hand a lid to cover the pan and extinguish the flames immediately. It is important that the pan has a handler and has the capacity to hold triplethe quantity of sealant you want to prepare. To determine the quality of the sealant, it is enough to drop a little bit in a cold dish, an immediately you will see its degree of tenacity”.

According to the author “one of the greatest advantages of this sealant is that it can be prepared aboard ships and can be used immediately after the fishes or molluscs have been collected”. In order to apply the “lithocolla”, the authors suggested.

“After the cork stoppers were adjusted in the jar mouths and having dried them well with a dry cloth, to take all humidity, heat the sealant until it boils. Mix it well and with a coarse brush made of pieces of an old cloth, attached to the end of a rod, apply a layer of lithocolla on the surface of the stopper. Sometimes the sealant when penetrating the cork evaporates some of the wine spirit, which shows up on the surface. With this evaporation, small holes are formed, which are perfectly filled by applying a second layer of lithocolla, after the first layer has dried out. When the jars are small, it is enough to invert them and dip them in the pan with the lithocolla. Repeating this immersion two or three times, the layer thickens to the desired point. It is also useful to cover the jars sealed this way with a well-tightened cloth that can be covered with a liquid resin: and, in the larger jars, the cork stopper should be tied with a strong string that contours the jars, forming a cross over the stopper. The jars prepared this way can, without any inconvenience, be turned upside down, be exposed to the agitation of storms and handle the strongest of the heats, without the liquor evaporating”.

These instructions must have served as the basis for the first collections assembled on the then recently established Natural History Museum of Rio de Janeiro, Brazil which is currently the Museu Nacional da Universidade Federal do Rio de Janeiro (MN/UFRJ). It is unknown if any fluid preserved specimens from such early times still exist in the MN/UFRJ collections and if these instructions were used and applied by naturalists, based in metropolitan Portugal. Brazil would become independent from Portugal on 7 September 1822, three years after the publication of these instructions, leading to an overall separation of both countries, not only politically, but also in terms of scientific practices.

The following decades in Portugal continued to be marked by serious turmoil. Still recovering from the impact of the Napoleonic invasions, Portugal would enter into a bloody civil war that lasted from 1832 to 1834. This war had major effects in the Portuguese society, including its scientific and natural history institutions (Ceríaco 2021). The outcome of the civil war marked the end of the former absolutist monarchy and the birth of a liberal monarchy, which brought deep changes to the Portuguese society. For natural history institutions, perhaps the most notable change was the closing of the former Royal Cabinet of Ajuda and the establishment of a new National Museum in the premises of the Lisbon Academy of Sciences in 1836. All the collections held in Ajuda, as well as its staff, were relocated to this new museological institution during that year. The “new” museum not only inherited the Ajuda collections, but also some others from the extinct religious orders and other private collections. Francisco Assis de Carvalho (1789–1851) was entrusted with the direction of the museum and with following the tradition of Ajuda and the Academy; he penned and published a new set of instructions to be distributed throughout the Portuguese overseas territories, as well as mainland Portugal. These were published in 1836 under the long title “Instrucções sobre o modo de preparar, e conservar accidentalmente os differentes exemplares zoológicos, que houverem de ser conduzidos das possessões Portuguezas ultramarinas até à sua definitiva preservação” [translation: Instructions on the methods of preparing and incidentally preserving the different zoological specimens that should be shipped from the overseas Portuguese colonies until its definitive preservation] (Carvalho 1836; Fig. 5). Consisting of 11 chapters, the instructions were presented taxonomically by class: mammals (chapter 1), birds (chapter 2), amphibians (chapter 3), turtles (chapter 3 [sic]), lizards, frogs and snakes (chapter 4), fishes (chapter 5), insects (chapter 7), testaceans (chapter 8), arachnids (chapter 9), zoophytes (chapter 10) and larvae, cocoons and chrysalides (chapter 11).

Regarding mammal specimens, fluid preservation could be used in different situations. If a larger animal was skinned, the skin could be preserved in wine spirit after being prepared and, for that, it would be immersed in wine spirit with a concentration between 14° and 18° Baumé. If the skin was too humid or still contained much greasy substances, the concentration could be higher by one degree or the fluid substituted. The same process would be used for the whole specimens of smaller animals, which should be eviscerated first. In the case that the specimens were too large, some parts of the specimens could be separated and preserved in fluid, such as the heart, the lungs, the stomach or even the vertebral column, which should be placed in a labelled jar filled with wine spirit at 18° Baumé. Whole birds could also be preserved in wine spirit and, as Carvalho noted “we saw in the Ajuda Museum perfectly prepared specimens by a black servant, the specimens had arrived in an infusion of wine spirit, before being prepared”. For turtles, lizards, frogs and snakes “that are not of an extraordinary size”, they should be preserved in wine spirit at 20°, being previously cleaned/washed. Some specific notes were provided for “Dragons” (species of the genus Draco) and venomous snakes. “Dragons” should be preserved in wine spirit (Carvalho did not note the concentration) and venomous snakes should come as whole specimens preserved in wine spirit between 20° and 24° Beaumé. It is interesting to note that the use of wine spirit to preserve venomous snakes as whole specimens instead of skinning them, rested on the fact that Carvalho was worried that naturalists could stab themselves with the venomous fangs of these animals. Small specimens of fish should be preserved in wine spirit between 20° and 22° Beaumé. For insects, Carvalho notes that only those with elytra (such as coleopterans) should be preserved in fluid (wine spirit at 14°) for shipment, being then re-prepared dry after arrival in the museum. In the mollusc realm, regarding fluid preservation, Carvalho only provides instructions for bivalves, which should be preserved in wine spirit (concentration not provided). According to the author, this would be the only way to conserve them, but noting that, in the case of shell-less molluscs, such as slugs, the preservation of the animal shape was problematic even with wine spirit. According to Caravalho, one previous curator of the Ajuda Cabinet, Félix de Avelar Brotero (1744–1828) suggested that the best way to kill and preserve these molluscs would be to toss them into strong wine spirit, but Carvalho considered that it would be desirable to kill them in strong wine spirit, but then transfer them to a lighter spirit for preservation. Arachnids should be preserved in wine spirit (14° to 18°). No fluid preservation was indicated for zoophytes, larvae, cocoons and chrysalides. The collections of the National Museum of Lisbon had a brief growth period after the publication of Carvalho’s instructions, but the situation of the Museum and its collections steadily deteriorated to the point that the Museum had to be closed to the public to avoid a “national shame” (Ceríaco 2021). No fluid preserved specimens prepared following Carvalho’s instructions are known to have survived to the present day.

On 8 January 1835, Joaquim José da Costa de Macedo (1777–1867), Perpetual Secretary to the Academy of Sciences wrote to Luis Mousinho de Albuquerque (1792–1846) at the time mayor of Funchal, in Madeira Island, asking for a collection of molluscs and zoophytes from Madeira Island. Following the request, were a few lines containing instructions on how these specimens could be collected, preserved and shipped. Macedo (1835) requested that two specimens of any collected species should always be shipped and that molluscs, if possible, would be preserved in wine spirit. More than a decade later, Macedo would issue a new set of instructions, this time directed to the President of the Council for Public Health, Francisco Ignacio dos Santos da Cruz on 28 May 1849, entitled “Breves Instrucções sobre a maneira de colher conchas, búzios, ouriços, estrelas, e plantas marítimas, para a colleção do productos naturaes de Portugal do Museu da Academia Real das Sciencias de Lisboa” [translation: Brief instructions of the methods of collecting shells, snails, sea urchins, star fishes and marine plants for the collections of Portuguese natural products of the Museum of the Royal Academy of Sciences of Lisbon] (Macedo 1849). These instructions were mostly focused on bivalves and shelled gastropods, sea urchins, sea stars and algae and aimed to complete the Portuguese collections of the museum. Surprisingly, no reference to the use of any kind of fluid preservation appeared in the instructions from Macedo’s (1849) instructions.

By the end of the first half of the 19^th century, the council of the Faculty of Philosophy of the Universidade de Coimbra, at the time in charge of the Natural History Museum of the University, notified the Queen about the status and needs of the Museum (Carvalho 1872; Ceríaco 2021). After completing an inventory of the Museum, the academic authorities recognised that its collections were considerably poor and needed more specimens. To enrich the collections, José Maria de Abreu (1818–1871), at the time Professor of the Faculty of Philosophy of the University of Coimbra, was entrusted to write a set of instructions to be distributed to the metropolitan and colonial authorities, as well as to diplomatic agents in foreign countries. These were issued in 30 July 1849 under the title “Instrucções para a colheita, preparações, acondicionamento e transporte dos productos e exemplares dos três reinos naturaes” [translation: Instructions to collect, prepare, house and ship the products and specimens of the three nature kingdoms] (Abreu in Carvalho 1872). Abreu’s instructions were much less detailed than those issued by Carvalho (1836) and, for what fluid preservation is concerned, it only mentioned that “small and soft animals, such as caterpillars, vermin, spiders etc. are immersed in simple or camphored wine spirit. The wine spirit (aguardente) must be at least of 30° of Beaumé”. Mammals should be deposited in a barrel full of wine spirit, which should be changed before the trip. The same should be done for birds, reptiles and fishes, but the latter two should be well-cleaned and dried before being immersed in the spirit. No instructions for fluid preservation of invertebrates were provided. It is unknown if these instructions achieved their objectives and it is difficult to pinpoint any specific specimen currently in the collections of the Coimbra Museum that has been prepared following this set of instructions.

In 1858, the collections of the National Museum of Lisbon held in the facilities of the Academy of Sciences were transferred to what would become mostly a “new” museum in the premises of the Polytechnical School of Lisbon (Ceríaco 2021). The new museums would be divided into different thematic sections, with the newly-created “Zoological Section” being entrusted to the direction of the Portuguese zoologist José Vicente Barbosa du Bocage (1823–1907). With Bocage as Director of the Zoological Section, the collections grew in number and scientific importance, turning what was an almost abandoned collection into one of the most scientifically prolific natural history museums in Europe (Ceríaco 2021). Aiming to enrich the collections of the Museum, Bocage published a new set of instructions in Portuguese, especially aimed at the Portuguese colonial officers and the educated civil society living and working in the overseas territories (Bocage 1862; Fig. 6). This new set of instructions was considerably disseminated throughout the Portuguese empire and its influence marked the ensuing decades.

Contrary to the previous sets of instructions, Bocage (1862) provided information on the methods and techniques that should be used to collect and kill different animals. For fishes, it was suggested that the specimen should be directly immersed in 36% ethanol, while for amphibians and reptiles, these should be shot with rifles, killed with spears and knives, collected with nets or directly by hand, or even hitting them in the back with a flexible rod so they would die, but not get too much damage. Birds should be shot with a rifle, while for mammals, Bocage suggested simply following the lead of the local hunters. For fixation, the author provided different formulas and techniques for the different taxonomic groups. For fishes, the preferred fixatives were wine spirit at 20° or Goadby liquor. The specimen should be first washed with cold water and a small incision was needed in the venter so the fixative could enter. Afterwards, the specimens should be wrapped in cloth or paper to avoid rubbing against other specimens in the can or jar used to transport them. For fishes, Bocage (1862) also mentions the use of the Ricord technique, which consisted of gutting the specimen and salting it under the sun for a three-day period, after which the specimen should be immersed in “aguardente de 18°” for two days and then transferred to an equal mix of “aguardente” and salt, which would constitute the final preservative. For herpetological specimens, the author suggested that the specimen, after being washed with running water, should then be immersed in wine spirit (20° to 22° (Beaumé)), let it rest for a couple of days and, after that, change it again to new wine spirit. The same was suggested for birds. For mammals, Bocage suggested that any specimen the size of a “hare or a small dog” should be sent as a whole specimen (i.e. not skinned) and, thus, preserved in fluid. For that, an incision should be made in the specimen abdomen so the fixative could enter and even inject the fixative through the mouth and anus with the help of a syringe. Skins could also be shipped immersed in fluid. The suggested fixative was wine spirit at 20° to 22° of the Beaumé scale, but Bocage also suggested a few alternatives in the case the latter was not available, namely the Goadby preserving fluids, for which he provided the following formulas:

1. Sal gemma [or normal salt] 125 grams (4 ounces)

Alum 65 grams (2 ounces)

Corrosive sublimate 1 decigrams (2 grains)

Boiling water 1 kilogram (2 lbs)

1. Sal gemma 250 grams (8 ounces)

Corrosive sublimate 1 decigram (2 grains)

Boiling water 1 kilogram (2 lbs)

Bocage advised the use of the latter, as according to him, the “alumen attacks the bones” and also that “whenever possible is convenient that the water is distilled”. He would still refer to the possibility of an additional method, originally suggested by “Mr. Rousseau, adjunct naturalist of the Paris Museum”, which consisted of a mix of 1l parts of water with two grams of creosote. This method, according to Bocage “has regarding alcohol or wine spirit, the advantage of not attacking the colour or contracting the soft tissues”. Bocage would still advise that the specimen “should be kept in that fluid for ten to fifteen days and then the fluid replaced for a fresh mix, with the same composition and strength” before being shipped to the museum. For the specimens to be shipped to the museum, they should be placed in either glass jars or wooden barrels, as filled as possible with the same fluid used for fixation and well closed, potentially using tar and cloths.

Regarding invertebrates, Bocage’s set of instructions covered the major groups, such as molluscs, crustaceans, insects (including their larvae), arachnids and myriapods (which included centipedes, “vermin” and “zoophytes”). Starting with molluscs, the instructions noted that they should be killed by drowning them in saltwater in a completely filled jar or, alternatively, by immersing them in water with some vinegar. In the case of univalves, the tip of the shell or spire should be broken so the animal would be in contact with the fluid, while bivalves and any other “naked” mollusc would not need any previous operation before being fixed. For fixing, Bocage suggested wine spirit at 18° to 20° Beaumé, noting, however, that the wine spirit, besides expensive, would shrink the specimens and discolour them. As alternatives, for “naked molluscs”, Bocage suggested suggested the formula:

Sal gemma (chlorureto de sodium) 125 grams

Alumen 65 grams

Mercury (II) Chloride 1 decigram

Water 1 litre

For molluscs with a shell, the formula suggested was:

Sal gemma (chlorureto de sodium) 250 grams

Mercury (II) Chloride 1 decigram

Water 1 litre

Alternatively, Bocage suggested the creosote formula (2 grams of creosote per litre of water). The instructions were also quite adamant that the fluid should be substituted after eight days. For crustaceans, which Bocage advised should all be sent preserved in fluid, the suggested fixatives would be wine spirit at 22° Beaumé or a solution of salt without alumen. Regarding insects, except those whose fluid preservation was unadvisable such as butterflies, Bocage advised the preservation in alcohol of 24° to 25° Beaumé, especially those that had a softer body that would easily rot or deform if dried, such as praying-mantis or grasshopper. The author also advised that, for specimens that would be preserved dry, but which had a “voluminous” abdomen and that the colours would not be affected by the alcohol, the specimen could be immersed for a few days before being pinned to their respective boxes. Butterfly larvae should also be preserved in fluid, either in wine spirit (which would discolour them) or in a solution of 2 g of creosote for one litre of water. Arachnids were, according to Bocage, problematic as no solution had yet been found that would adequately conserve them: dried and pinned to boxes would considerably deform them, while in alcohol, they would lose their colours. Due to that, Bocage suggested preserving them both ways (without providing any specific alcohol concentration), but also using the creosote formula mentioned above. For myriapods, the instructions divided them into three main groups. For the “true” myriapods (centipedes etc.), Bocage refers that specimens should be preserved in the same type of fluid preservatives already mentioned to the other animals. For “vermin”, they should be either preserved in alcohol, creosote solution or the same formula suggested for “naked” molluscs. Finally, the softer “zoophytes” would also be preserved in the same type of fluids already mentioned for other animals (without mentioning any specific formula). For shipment, the molluscs, crustaceans and insects should be placed in adequately sized vials and conditioned in a way that would not leak. For all the specimens, the fluid should be replaced before shipping.

Bocage’s instructions were considerably distributed and resulted in the shipment of thousands of specimens to the museum. This was especially due to the diligence of several collaborators and explorers hired by the museum to collect in Portugal and its overseas territories, namely the explorer José de Anchieta (1832–1897) and Francisco Newton (1864–1909) in the Gulf of Guinea, Cape Verde and Guinea Bissau (Ceríaco 2021). Surviving early 20^th-century catalogues show that the collections of amphibians, reptiles, fishes, but also mammals were considerably large, reaching several thousand specimens, mostly preserved in fluid (Fig. 7).

Unfortunately, most of these specimens were lost in the fire that engulfed the Lisbon Museum in 1978 and which destroyed its zoological collections almost completely. The few specimens that survived this fire were those that were outside the museum premises due to various reasons, but also the subset of specimens that were donated in the end of the 19^th century/early 20^th century to other museums in Portugal and Europe. Ceríaco (2021) listed the European and Portuguese institutions that received duplicate specimens from the Lisbon Museum at this time. Many specimens from Bocage’s time are still extant in large European Natural History Museums, such as the Natural History Museum (NHM) in London, the Muséum national d’Histoire naturelle (MNHN) in Paris or the Museum für Naturkunde (ZMB) in Berlin (authors pers. obs.). In Portugal, an important collection of African mammals, as well as a few Portuguese mammals and a miscellanea of amphibians and reptiles, was sent to the Zoology Museum of the Polytechnical Academy of Porto in 1908 (Ceríaco 2021). Many of these specimens are still extant in the MHNC-UP collections, such as syntypes of African mammals collected by José de Anchieta in Angola (authors pers. obs.; Fig. 8) or a rare specimen of the extinct Cabo Verde giant-skink, Chioninia coctei (Duméril & Bibron, 1839), collected by Francisco Frederico Hopffer (1828–1919) in 1889 (Ceríaco 2015; Fig. 9). Similarly, MCUC also has a few specimens of both amphibians and reptiles donated by the Lisbon Museum (Themido 1941; Ceríaco 2021). The few specimens that survived the 1978 fire and are still extant in MUHNAC are a small set of five specimens of Alytes obstetricans lusitanicus Ambu, Martínez-Solano & Dufresnes, 2024 (Fig. 10) collected by José Maria da Rosa de Carvalho (1817–date of death unknown) in 1860s, as well as a small collection of Portuguese and African fishes collected mostly by Francisco Newton between the 1880s and 1900s.

While rare, there were several amateur naturalists in Portugal during the second half of the 19^th century. Perhaps one of the most active, at least in terms of publication records, was the Porto-based amateur naturalist Eduardo Sequeira (1861–1914). Sequeira was especially interested in botany and herpetology. Interestingly, Sequeira would also publish a naturalist manual entitled “Guia do naturalista colleccionador, preparador e conservador” [translation: Guide for the collector, preparator and conservator naturalist], which would end up having two editions (Sequeira 1887, 1888). Sequeira’s manual was mostly aimed at other amateurs who wanted to assemble their private collections, especially those who were focused on taxidermic methods. However, it also provided several instructions on how to preserve different types of animals in fluids. According to the author, fluid preservation was particularly useful for research specimens, especially “those vertebrates of smaller dimensions which we want to further properly study”. As an overall preparation method, Sequeira (1887, 1888) suggested that “before immersing the specimens in alcohol, alcohol should be injected through all natural orifices and a small incision made on the venter so the spirits can rapidly enter the body and prevent the interior corruption”. While for mammals and birds, the author exclusively focused on taxidermic methods; for amphibians and reptiles, Sequeira notes that fluid preservation was preferable to taxidermy because “not only conserves better its colours, but also because it allows future dissection”. For that, one should use “wine spirit with a little quantity of water to diminish its strength” and the specimen should be washed after being killed, dried with a cloth and then tied through the neck with a string that would be tied to the stopper “to be easier to extract the specimen when needed”. The stopper “should be of glass; the air stopper should not be tightened with common wax, which does not prevent the evaporation of the alcohol, but rather with thick Arabic gum or with the following formula, generally used by all naturalists: yellow wax, normal resin (“breu dos marinheiros”), powdered red “oca (iron oxide) and turpentine”. To prepare this formula, Sequeira noted that “the quantity of each substance is measured according to the need of the formula to be more or less dense; the wax is melted together with the resin and, when everything is liquified, the oca is added, mix well and let it boil. After boiling for five to ten minutes, add the turpentine and keep it cooking, the density is evaluated”. This mix would then solidify and, whenever required to be used, it just needs to be boiled again and applied with a brush in the jars. For fishes, the same methods should be used or, instead of the alcohol solution, a solution made of “1 litre of water, 115 grams of common salt, 100 grams of alum and 12 grams of corrosive sublimated” could be used. Some insects, namely the caterpillars, should also be preserved in alcohol. The same applied to myriapods, “vermin”, crustaceans and arachnids. The latter should be preserved in small tubes, guarded from the sun in appropriate boxes (Fig. 11). For molluscs, the preservation solution should be wine spirit at 18° to 20° Baumé and, for the mollusks without a shell, it could also be a formula of “125 gr pure salt (sodium chlorite), 65 gr of Alum, 1 dg of mercury (II) chloride and 1 litre of water”. For the molluscs with external or internal shell, the formula would be “250 gr of pure salt (sodium chlorite), 0.1 gr of mercury (II) chloride and 1 litre of water”. For sea-urchin and starfish, Sequeira suggested preserving them in “very weak alcohol in which a little bit of salt is dissolved or in ether, sodium cyanide, benzine, phenolic acid, a mixture of alcohol with boric acid, glycerine and creosote and colourless sugar infusions in alcohol. The only difference between the first and the second edition is that the latter had a section about “Special Collections” where some additional notes were made about the collection of bones and organs. While we assume that Sequeira had his own collection, we have no information about it and what happened to it after Sequeira’s death and no specimens seem to have survived.

Bocage and Sequeira’s instructions for fluid preservation were published a few years after the invention/discovery of formaldehyde in 1859 by the Russian chemist Aleksandr Butlerov (1828–1886). While formaldehyde solution would become the most used fixative for zoological collections until the present day, its wide application for fixation of zoological specimens would only become popular at the end of the century (Moore 1999; Simmons 2014). In Portugal, the first references to the use and application of formaldehyde as fixative for zoological specimens were published in 1895 by two zoologists associated to the Museu de Zoologia da Universidade de Coimbra, Adriano Xavier Lopes Vieira (1841–1910), at the time Director of the Museum and Manuel Paulino de Oliveira (1837–1899), at the time naturalist of the same Museum (Fig. 12).

In a paper about the conservation of zoological specimens, Vieira (1895) refers the problems associated with the preservation of fishes or any other animals preserved in alcohol, which, in his opinion has “the serious problem of deformation through the retraction that it implies in the tissues of all soft animals and of discolouring all animals, probably with the exception of mammals”. Vieira (1895) continues noting that the alcohol preservation was “very expensive, due to the need for glass containers, the cost of alcohol and its constant evaporation”. Vieira suggested the replacement of alcohol for other preservative fluid, namely a “solution of formol at 4 × 100”, that he assumed is “very advantageous, due to its lower discolouration power, fast hardening action, without much retraction or deformation of the tissues and its cheap price”.

Immediately after these considerations about the pros and cons of alcohol and formaldehyde, Vieira provides an interesting account on the way to present specimens inside the jars, but also on how to seal them. Vieira notes that he had made a “considerable progress with the system of fixation of small animals to glass slides added inside the jar with the preservative, in which the specimens are maintained in the same plane in order to avoid deformation which, when free or floating, suffer against the walls of the cylindric jars”. This new “system” would also avoid the need of a glass fluctuator, a glass “bubble” attached to the specimen or any attaching thread, both of which could also cause problems: the fluctuators, besides spontaneously breaking inside the jars, would also descend if the alcohol level dropped, allowing the specimen to sit in the bottom of the jar, while the attaching threads would allow the escape of the alcohol outside. Despite the discussion and praise of this system, Vieira did not provide any details of what it consisted.

Vieira (1895) was, however, more enthusiastic about what he called “emzéd cement, a mixture of 2½ parts of paraffin and 1 part of natural rubber”, which was “applied hot on the mouth of the glass jar, to glue the closing glasses, which are slightly warmed in a flame”. The author highly praised “the inventor” of this cement (although never identifying him”, as “before it we only had, to resist alcohol [evaporation], the top of glass bocals, the glassmaker paste or mixture gum with some inert powder such as calcium carbonate”, but “the glassmaker mass solidifies too much and does not allow opening the glasses, as it is so many times necessary, with risking breaking the tops, even if it does not allow the evaporation of alcohol (…)” and the “the massa de goma gets attacked by the condensate alcoholic vapours and cracks, allowing the vapours of the liquid to exit”. In this sense, according to Vieira (1895), the “emzéd cement” was the perfect solution, as it did not “crack, does not solidify too much, does not allow the evaporation of more than a small percentage of the alcohol, glues perfectly and can be unglued “cold”, anytime we want”.

Oliveira (1895) presented the first Portuguese case study on the use of formaldehyde to euthanise and fix marine invertebrates. In a paper entitled “Préparation et conservation de quelques animaix par l’aldéhyde formique”, Oliveira presents the context, methods and main results of his experimentation with this fixative. Oliveira, at the time Professor in charge of the zoology course at the Universidade de Coimbra, wanted to study the tunicates, nudibranchs, coelenterates and other marine invertebrates of Portugal, but faced difficulties with the available fixation and preservation methods he had been using. According to Oliveira, these methods “deformed or changed the natural positions” of the specimens, bleached the specimens, retracted and tensed the body of the specimens, hardened the specimens in a way that prevented their preservation in natural positions, made transparent animals opaque and altered the cellular structure and consequently affecting any histological studies. Oliveira contacted the German naturalist Felix Anton Dohrn (1840–1909) from Naples Zoological Station, from which Oliveira had previously received marine invertebrate specimens that were better preserved than those he initially attempted to preserve. The intent of Oliveira was to ask Dohrn for the technique, but Dohrn wanted to keep it secret until he could publish it, which happened in a paper published in the Mitteilungen aus der Zoologischen Station zu Neapel (Lo Bianco 1890). This paper would be translated into Portuguese and republished by the Portuguese ichthyologist and naturalist of the Lisbon Museum Balthasar Osório (1855–1926; Lo Bianco (1892)). After experimenting with methods suggested by Dohrn in his paper, Oliveira was not happy with the results, as specimens would lose their natural positions and colours. In 1894, Oliveira read the paper by Cohn (1894) entitled “L’ aldehyde formique et son action sur les bactéries” and published in the journal “Annales agronomiques”. As formic aldehyde was not available for purchase in Portugal, Oliveira ordered it from abroad and received a 40% solution under the name “formol” (which he called “solution de formol” or “solution normale” – according to J. Simmons, pers. comm., formol was a German trade name for the commercial product of aqueous formaldehyde, a 40% solution was a saturated solution of formaldehyde gas in water), which he then diluted using “4 volumes of formol for 100 volumes of water”. He would then try both the formol and its 10% dilution of the formol (the equivalent of a 4% dilution of formaldehyde) with several different types of marine invertebrates, such as anemonae of the genera Anemonia, Actinia, Bunodes and Corynactis, crustaceans of the genera Palaemon and Anchistia, as well as some salpa. Using some drops of the “solution de formol” on the different tanks filled with sea water and containing the specimens, Oliveira saw that the specimens would die in a few minutes without retracting their appendages and thus keeping the much desired “natural position” and they would also keep their original colouration. Oliveira then proceeded to test the reaction of colours when the specimen was transferred to alcohol at “25 degrees” after being fixed in formol for eight days. In the two cases, there was absolutely no change of colour in the specimen nor in the alcohol after the transfer, while in other species, the alcohol initially became stained brown, but after being replaced once, both the specimen and the alcohol maintained their original colours. Oliveira noted that there were no significant differences between using the “solution normal” or the 10% dilution, noting that the latter would probably be better for specimens destined to be used in histological studies. After 15 days, both the specimens fixed and preserved in formol and those fixed in formol and later transferred to alcohol maintained their original colours, leading Oliveira to believe that this would be indefinitely preserved in either situation. Not having any fresh specimen of salpa available, Oliveira tested the preservation of the transparency of these animals by immersing some specimens that had been previously preserved in Owen’s solution. As the specimens remained transparent, Oliveira assumed that formol would also be suitable for these situations. Given the results of his experiences, Oliveira drew six major conclusions regarding the application of formol: “1^st It is convenient to kill the species used and probably others, without them losing their natural positions; 2^nd To preserve the colouration of the animals, we prefer it to any other technique tried. We denote that the difference between these specimens prepared with this substance and those obtained through other techniques is between life and death. These results are sustained by the observation [of the colours] after 15 days and we believe that the colours will be preserved in the solution de formol or in the alcohol after its immersion for a few days in the solution normale; 3^rd The produced hardening is quite considerable to the conservation of the natural positions; 4^th It does not produce a very noticeable shrinkage; 5^th We tend to believe that it will conserve the transparency of those animals that possess this characteristic; 6^th In a solution of formol diluted at 10%, the hardening is compatible with histological studies”.

Interestingly and despite his enthusiasm towards formol, for amphibians and reptiles, Oliveira continued advocating the use of alcohol as a fixative (and preservative). In an important and long-lasting contribution to the study of Portuguese herpetofauna (it had three editions: Oliveira (1896; 1908; 1931), Oliveira suggested the use of jars with alcohol of “30° Cartier, more or less” to preserve amphibians and reptiles (Oliveira 1896; 1908; 1931). The author suggested that the specimen should be immersed in alcohol in a “natural position” and, to achieve that, after being euthanised “one or more incisions should be done in the medium abdomen line” and laid in cork or wooden sheets, pinned by their members in the desired position. The specimen laid in these sheets should be left for a few days in alcohol to “harden” and only then should be taken out of the sheets and transferred to the definitive jars. To properly display the specimen in the jars, Oliveira refers to a method adopted by his colleague Lopes Vieira in the Coimbra Museum, where the specimens would be tied to transparent and colourless glass plates with a thin thread. This way, according to the author, it simulated that “the specimen is suspended in the fluid and can be examined by all sides, not being necessary to open the jars unless when the disposition of the teeth, the shape of the tongue etc. needs to be studied”. For sealing the jars, Oliveira suggested the “Emzed cement” and provided a detailed description of its preparation and application process:

Preparation: Take two to three parts of paraffin in weight and one of natural rubber divided into small pieces, throwing them in a metallic pan that is put to the fire until [the mix] melts. Take and conserve the coagulated mix.

Application: Slightly heat the cement until it melts and apply it with a brush in the mouth of the jar that you want to seal. After this, heat the glass pieces that will be used as a lid in a flame and, when hot, place them over the already coagulated cement in the mouth of the jar. The temperature of the glass piece melts the cement again and it attaches the lid to the mouth [of the jar].

Process to open the jars: Generally, it is sufficient to use a pocket knife to open them. However, when needed, to avoid the lid being broken, which unfortunately happens, drop hot water over it to soften the cement, which easily softens and lets the lid “unglue” itself.

According to the author, this sealant “is not only useful for reptiles and amphibians, but also for other animals” and had worked very well for the Coimbra team. Oliveira recalled the moment when “recently we transported from Algarve to Coimbra large tin cans sealed with this cement, which contained different types of animals in alcohol and they arrived perfectly fine”. Lastly, Oliveira noted the importance of recording the colour of the specimen, namely the “greens, yellows and reds”, which could be altered by the alcohol, as well as the “colour and shape of the iris”. Coimbra’s fluid-preserved specimens are still mostly extant in the collections of MCUC, partly in the historical exhibition rooms (Fig. 13) and other parts in collection rooms.

While Bocage’s (1862) instructions certainly had an outstanding importance in the first decades of the second half of the 19^th century, especially for overseas collectors, Vieira’s (1895) and Oliveira’s (1895) papers were surely influential to a new generation of Portuguese museum naturalists. It is important to note that both Vieira’s (1895) and Oliveira’s (1895) papers were published in the journal “Annaes de Sciencias Naturaes”, whose editorial board included the young zoologist Augusto Nobre (1865–1946) who would later become responsible for establishing what would become the Zoology Museum of the Polytechnical Academy of Porto, currently part of MHNC-UP. In fact, besides the aforementioned specimens and collections in MCUC, some of the late 19^th-century/early 20^th century vertebrate and invertebrate specimens of the Portuguese fauna currently housed in MHNC-UP have been fixed, preserved and displayed using these methods (pers. obs.).

As the naturalist of the new Zoological Museum at the Polytechnic Academy of Porto and entrusted with the establishment of a comparative anatomy class, Nobre prepared a textbook for the classes, entitled “Estudos de Zootomia” [translation: Studies of Zootomy] (Nobre 1892). The book would have a second edition (Nobre 1894), mostly identical to the first one. The textbook was mostly directed to the practical classes of comparative anatomy, where the students were directed to dissect specimens, prepare tissue slides and compare the different anatomical parts of a wide range of animal groups and not much to the preparation of specimens to be deposited in collections. Nevertheless, Nobre (1892, 1894) still provided a list of the most common preservation fluids in anatomical studies, which were:

“Alcohol – this is the fluid that presents better results to the preservation of animals. Initially placed in alcohol at 60°, then they are changed to another alcohol at 70° or 75°. The specimens preserved in alcohol are mostly for the study of the nervous system, the other systems deforming themselves too much or even entirely. If not for the study of the nervous system, it is preferable to use fresh animals.

Acetic acid – it is used to make the nervous system visible. It also makes the muscular and conjunctive tissues clearer.

Chromic acid – is rarely used in anatomy. The nerves when immersed in this fluid become more visible under a microscope. The dilution in water is either 1/100 or 1/1000.

Picric-sulphuric acid – It is good for the study of coelenterates. It hardens the soft tissues facilitating dissection. The coelenterates, after being killed with chloral hydrate, are immersed in this fluid; and after that are immersed in alcohol for long-term preservation or used for dissection. Without the use of these fluids, it is almost impossible to preserve these animals in alcohol, because their tissue destroys itself. The prepared mixing requires a saturated solution of picric acid with two parts of sulphuric acid, adding afterwards two to three times its volume in water. The solution is decanted, separating the precipitate. This process to prepare coelenterates is still poorly known. It is used in the Naples Zoological Station” (Nobre 1892, 1894).

Back in Lisbon, the Royal family was also personally involved in natural history studies. King Carlos de Bragança (1863–1908), also known as Carlos I of Portugal, was not only a strong supporter of natural sciences, but he was himself a zoologist who organised and actively participated in oceanographic expeditions, where he collected specimens that he later described and published (see Ceríaco (2021) for an overview). The King had two museums within the Royal Palace of Necessidades – one dedicated to birds and mammals and the other to oceanographic collections (Fig. 14). Due to his strong passion for oceanography and close collaboration with his also oceanographer cousin, King Albert of Monaco (1848–1922), in the late 19^th/early 20^th century, King Carlos organised several oceanographic expeditions off the coast of Portugal, where he collected several thousands of fishes and marine invertebrates. The King wrote and published a report of the first expeditions (from 1896 to 1900) and provided an interesting and detailed chapter on the fixation and preservation methods employed both to vertebrate and invertebrate specimens (Bragança 1902).

According to this report, the specimens were prepared in a small laboratory in Cascais, to where all the collected specimens were sent, still alive, immediately after the end of each expedition. According to the King, fishes were generally conserved in alcohol at “27° Beaumé (70° Gay-Lussac)”, but for more fragile abyssal species, alcohol was only applied after an initial fixation with “4% formol”. To conserve the specimen colours, the specimen was first “gently washed in freshwater”, an incision was made and the specimen was immersed in a solution of “4% formol” for about an hour. After that, the specimen was once again washed with fresh water and then put in a solution of “glycérine formolée”, which was prepared following these steps:

“To 700 cm³ of common commercial glycerine, at a concentration of 28° Beaumé (° = 1,240), we add 200 cm³ of distilled water, which will produce a mixture with a concentration of 23° Beaumé (° = 1,1290). We leave it to rest for 24 hours, to completely eliminate the air bubbles, then we add 20 g of formaldehyde and mix it with a glass wand”.

According to the author, the solution “conserves itself for a long time, but it is a good idea to renew the formol when it starts losing its characteristic odour”. Whenever these specimens needed to be studied at the lab, glycerine could easily be cleaned from the specimens by washing it in pure water. For invertebrates, the author refers that these were conserved in alcohol at the concentration of “25° Beaumé (64 Gay-Lussac)” after a fixation in a solution of 4% formol or after the “methods used in the Zoological Station of Naples”. However, Carlos I noted that he also used some “personal methods” that had provided him better results, for the “Hydromedusas” and the “Anthozoans”. These personal methods consisted of: “After letting them [the animals] spread their parts in large jars or in aquariums (in the laboratory), they are quickly immersed, without washing, in a very strong solution of formalin (10%). They are left there for about an hour for fixing, then washed in pure water and then preserved in weak alcohol 21° Beaumé (53° Gay-Lussac) or in the 4% formalin solution”. According to the authors’ own words, following this procedure, he had “obtained several specimens that were perfectly spread and expanded” which if instead of being preserved in alcohol they had been preserved in the “glycérine formolée”, they would “certainly keep their colours”, as was the case of some specimens of bathypelagic medusas that the King himself had prepared and preserved for three years. Crustaceans would be fixed in 4% formol and preserved in “glycérine formolée” and this way, species with intense colours such as “Gnathophausia would indefinitely preserve their colours”. To attest his claims, the report was illustrated by plates depicting different marine invertebrates of the King’s collection (Fig. 15). During the twentieth century and especially after King Carlos death in 1908, the collection experienced a complex path of dispersal until it was finally deposited in the AVG, where a good number of these specimens are still extant today (Fig. 16).

Recalling the beginning of the 19^th century, the beginning of the new century was also marked by social and political instability in the country, which would again impact on the natural history museums. Bocage, who had become the most respected Portuguese zoologist of his time and in 1905 had the zoological section of the National Museum of Lisbon renamed as Museu Bocage after him, died in 1907. In 1908 King Carlos I and his older son Luis were assassinated in Lisbon and in 1910, the Portuguese monarchy was overthrown and a republican system implanted. The following years, Portugal, as the rest of Europe, participated in World War I (1914–1918) and suffered the dire consequences of the Spanish Flu pandemic (1918–1920). These disrupting events, together with the political and social turmoil that affected the early days of the new regime, eventually led to the end of what would be called the First Portuguese Republic in 1926, which was replaced by a military dictatorship. In this difficult context, Portuguese scientific institutions faced difficult times.

Apart from the new edition of Oliveira’s work on Portuguese herpetofauna (Oliveira 1908), the first naturalist instructions published in Portugal at the beginning of the century were those issued by the Sociedade de Geografia de Lisboa in 1912 (Anonymous 1912). The Sociedade de Geografia de Lisboa was a private scientific society established in 1875 to promote geographic studies in the Portuguese world, with a main focus on the then-Portuguese colonies in Africa and Asia. Since its beginning the Society had a small museum, mostly dedicated to ethnography and historical material associated with the Portuguese empire, but also a small natural history collection. Its instructions, entitled “Instruccções abreviadas sobre a collecção e remessa de productos zoológicos e coloniaes” [translation: Abbreviated instructions about the collection and shipment of zoological and colonial products] were printed in Lourenço Marques (current day Maputo), the capital of the then-Portuguese colony of Mozambique (Anonymous 1912). As with most of the 18^th and 19^th-century instructions, this new set followed the traditional taxonomic division, starting with mammals and continuing to birds, amphibians and reptiles, fishes and invertebrates. For mammals, fluid preservation was only suggested for small specimens and foetuses. There was no indication of what alcohol or concentration of it should be used, but simply that the specimens needed to have “a deep incision in the venter, without damaging the intestines, so the fluid can penetrate the whole specimen. When this is not convenient, the fluid should be injected through the mouth or the anus with a syringe”. The specimen should then be immersed in the fluid. For birds, not much detail was given regarding fluid preservation, the authors only noting that “fluid preservation is preferable in circumstances when there is the lack of time and materials to prepare the skins” or when the specimen was already in some state of putrefaction. For reptiles, there were more details, as fluid preservation was the preferred method noted by the authors. For that “any spirit or aguardente that is sufficiently strong” could be used and “an incision in the central part of the venter should be done so the alcohol penetrates in the interior of the body and the preservation is effective”. The authors also noted that “methyl alcohol can be used as it is cheap, but it only should be used to provisionally preserve the specimens and not be used to the rare and more curious specimens”. Overall, alcohol at 40° should be used. When there were no available jars, any other type of jar or tin container could be used. Specimens should be placed inside the jar, but with caution to avoid them being too tight against each other and then the container should be sealed, full of alcohol. Eggs should also be preserved in “strong alcohol”. For amphibians “due to the fragility of its cutaneous surface”, it was suggested to preserve them “in a less strong alcoholic fluid, to avoid the retraction of the skin”, but it would also be necessary to change the fluid two or three times and, in the end, replace it for alcohol “no less strong than 30°”. The same would apply to tadpoles and juveniles and, to ship them, they should be wrapped in cloth to avoid damage. The author would suggest the same processes for fishes, noting that saltwater fishes should be washed first with salt water and that because “fishes contain much more water than amphibians and reptiles, they dilute the alcohol levels of the preservative, which thus needs to be replaced more times”. Specimens should have their venter open and small incisions in the side of the spine should also be made so the preservative would act on the body. For larger fishes, it would also be important to extract the liver and any fat from the body cavity and then the specimen should be wrapped in cloth for transportation. The author also warned that, in the case of use “of any additional toxic substance in the alcohol, the collector should warn the recipients of the specimen”. Dipterans, arachnids and myriapods should also be preserved in alcohol and the latter two should be initially placed in “not strong or diluted alcohol to avoid their hardening” and the next day transferred to stronger alcohol, inside small tubes, to avoid being damaged or tangled. Finally, for molluscs, the authors went into additional details, namely on how to narcotise and euthanise the specimen, which, for the case of gastropods and lamelli branches, should start with a narcotisation with sea water and some alcohol, gradually increasing the concentration to 70% or with chloral hydrate at 1%. Then the animals would be killed with a concentrated acetic solution, under the sun or with boiling corrosive sublimate. Cephalopods should be fixed in 70 or 100% alcohol, with the precaution of making an incision in the venter so it could reach the body cavities, but this fluid should be renewed after 24 hours. For these same specimens, after three days, they could be transferred to a solution of formol at 4 to 5%, “which better preserves its colouration”. The same process would be applied to sea urchins. A small collection of fluid preserved amphibians and reptiles from the Sociedade de Geografia de Lisboa is currently deposited in MUHNAC. Most of the specimens, however, do not possess any kind of label or associated data and it is, therefore, impossible to determine their collection dates and localities, being also difficult to confidently associate them with the issuing of these instructions.

In 1929, the zoologist José Bettencourt Ferreira (1866–date of death unknown), at the time an auxiliary professor in the Faculty of Sciences of the University of Porto, but formerly a museum naturalist working under the supervision of Barbosa du Bocage in Lisbon, presented an interesting communication in the Portuguese Society of Chemistry and Physics entitled “Líquidos de conservação para peças anatómicas e exemplars de botânica e zoologia” [translation: Preservation fluids for anatomical, botanical and zoological specimens] (Ferreira 1929). While the title of the talk referred to both anatomical, botanical and zoological specimens, the content of the manuscript was mostly indicated for the preparation of anatomical specimens. The manuscript was also mostly a dissertation rather than a guide or instructions manual. Ferreira (1929) made comments about what were the most used fixatives – “the alcohol or to be correct the alcohols, the sublimate, the formol, the sodium chloride, the potassium chloride, the picric acid, the thymol, the chloroform etc.” – and that these should be used depending on the future desired uses of the specimen. For example, if the objective were to avoid losing the colours, then it was better to use “those whose main agent is the formaldehyde, which will not stop the use of other substances, considered, in this point of view, due to its energic fixative and anti-putrid power. Those are, in first place, the sodium and potassium chlorides – very abundant in nature, easy to get and of easy manipulation, then the alcohols, which comprehend the glycerine and, in last place, the toxic agents, with a high microbicide power – the Mercury (II) Chloride, the formic aldehyde, the picric acid, potassium dichromate and the salicylic acid, amongst others”. Ferreira points to the Melnikoff-Rasvendenkoff method for the preservation of anatomical specimens, amongst others. However, Ferreira’s preferred method was the following:

“The specimen or anatomical piece is immersed in formalin solution at 4%, after being washed in salted water (artificial serum) at 7% for 48 hours. Then transfer it to alcohol at 90°, in which it will rest for about 24 hours, minimally 12 if these are small specimens or fragments of organs. For definitive preservation, the specimen or preparation shall be put again in formol and after a few days put again in alcohol” (Ferreira 1929).

Ferreira (1929) also noted that both alcohol and formalin had their problems, namely alcohol “is a very fast acting dehydrator, which considerably shrinks the specimens and preparations, stiffening them and taking their flexibility completely, impeding the examination of those that had stayed for a long time in it. The discolouration is complete due to the attack that the alcohol does to the pigments”, while formol “besides its toxic power and the irritating vapours that affects the mucous membranes and are particularly harmful for the eyes, has the capacity to excessively stiffen the objects delivered to its action, which become brittle or, after some time, start to disintegrate”. To address this, the author suggested that to “correct the problems and inconvenience” of those solutions, “salicylic acid, either in concentrate and warm, simple or associated with formol in small proportions” should be used. A solution of 2.5 g of salicylic acid per 1 litre of water, at 15°C, should be injected into the cavities and digestive canal of the specimen, which would then be immersed in the solution, but changing the solution three times after around six days. For definitive preservation, Ferreira advised adding small quantities of formol, which could “vary from a few drops to one or two grams per litre”. According to the author, “the specimens or anatomical pieces preserved in this medium maintained their form and natural proportions, as well as their colours, with little alterations, if they pass through this process while fresh. The use of concentrated salicylic acid is a sufficient guarantee when the preparation is fresh and waives the addition of any other preserving agent. The formol, in a small percentage, has mainly the objective of giving consistency to the object, which, under the action of the salicylic acid, keeps its natural sagging which allows its manipulation and long-term study”. Ferreira (1929) would state that some vertebrate anatomical pieces that were prepared using this method and were displayed in the zoology laboratory of the Faculty of Sciences of Porto attested his conclusion. These specimens, however, did not survive until the present (LMPC and MPM pers. obs.).

The military dictatorship in Portugal transitioned to a new form of autocratic and authoritarian regime that became known as the Estado Novo, which would rule the country from 1933 to 1974. Despite its authoritarian and repressive nature, the Estado Novo brought some political and social stability to the country, as well as investments in the country’s scientific institutions. The first decades of the new regime were marked by the organisation of several scientific congresses, investment in existing scientific institutions and museums, as well as the establishment of new scientific institutions.

In 1941, the first national congress of natural sciences was held in the Faculty of Sciences of the University of Lisbon, sharing the premises with Museu Bocage, a scientific establishment of the Faculty of Sciences. One of the presentations at the congress was that of Fernando Pedrosa Mendes (dates of birth and death unknown), naturalist of Museu Bocage and son of António Mendes (dates of birth and death unknown), taxidermist of the Museum since the last decades of the 19^th century. Mendes talk was published the next year in the congress annals under the title “Colheita e preparação preliminar de animais para o museu” [translation: Collecting and preliminary preparation of animals for the museum] (Mendes 1942), where the Museu Bocage naturalist provided an abridged, but fairly complete overview of the main collecting and preparation techniques for the different zoological groups. To what fluid preservation is considered, Mendes noted that small vertebrates could be preserved whole in fluid. For that, the author warned the readers to avoid eviscerating the specimen and simply injecting the specimens with alcohol in the body cavity and main muscular masses. For the case of mammals and birds, Mendes suggests that the mouth and anus should be clogged with cotton, to avoid soiling the fur and feathers of the birds with body fluids. After that, the specimens could be immersed in “not that strong alcohol”. Mendes does not provide a concentration of alcohol. More details were provided, however, for the case of invertebrates. Mendes resumed the main narcotisation, fixation and preservation methods of invertebrates in Table 1.

A few years later, in 1945, António de Barros Machado (1912–2002), then a high-school teacher associated with the Zoological Institute “Dr. Augusto Nobre” specialsing in cave biology, published a small paper with instructions for “the biological exploration of caves” (Machado 1945). There, Machado provides a list of the materials needed for the preservation of the collected specimens, with “tubes made of thick glass, with alcohol at 70°” and “tubes made of thick glass com F.A.A. solution, a fixative and preservative fluid of very common use to plants and lower animals” or with “Beauchamp fixative (for planarias)”. Machado also provided the F.A.A. and Beauchamp solutions formula which was respectively made of “100 cc of Alcohol at 50°, 6 ½ cc of commercial formol and 2 ½ cc of acetic glacial acid” and “6 parts of Alcohol at 90°, 3 parts of commercial formol and 1 part of acetic glacial acid”. Machado also suggested different approaches for the fixation and preservation of the different cave animals collected: for “fresh-water vermin (…) which are mostly planarias (…) are put in “Beauchamp fixative” for several hours (no more than 24); preserve them then in alcohol 70°”; for “terrestrial vermin (…) anaesthetise in water to which a little bit of alcohol is added and are fixed and preserved in F.A.A.”; both the “terrestrial crustaceans”, “aquatica crustaceans”, “aquatic insects”, “insect larvae”, “terrestrial insects (Collembola, Diplura and Thysanura)”, “arachnids” and “myriapods” were simply fixated and preserved in 70° alcohol. For “molluscs”, although they should be also fixed/preserved in 70° alcohol, they “should be previously anaesthetised”. Fishes, amphibians and reptiles would also be “preserved in 70° alcohol (or also, in a worst case, in 8% commercial formol)”. Finally, bats “should be anaesthetised with chloroform vapours and then preserved in 70° alcohol”, but its parasites (dipterans, fleas and acari) should be previously collected and preserved in 70° alcohol. Machado also suggests that the fungi could be collected “together with its substrate” and preserved using the F.A.A. solution.

In addition, in 1945, the Portuguese authorities issued a comprehensive plan for the scientific study of the Portuguese overseas territories in Africa (Anonymous 1945). The plan included several zoological surveys to be conducted in Portuguese colonies and the Portuguese zoologist Fernando Frade Viegas da Costa (1898–1983), mostly known as Fernando Frade, was appointed to lead said surveys. Frade had been a student and naturalist in Museu Bocage and was, respectively, nephew and cousin of António and Fernando Mendes. In the following decades, Frade would be responsible for leading zoological missions to the colonies of Guinea-Bissau, Angola, Mozambique and São Tomé and Príncipe.

In a report of the first of these missions, the one to Guinea-Bissau, Frade et al. (1946) provided an extensive and detailed description of all the collections, preparations and conservation techniques used during the expedition. While most mammals were prepared as skins and skulls, some groups “mainly bats and rats, were preserved in alcohol due to the lack of time for skinning them”. For reptiles, the authors referred to several techniques. While “crocodiles, pythons and large snakes are to be treated as mammals [i.e. skinned], but leaving the skull attached to the skin”, for all other reptiles, an incision was made into the abdomen “respecting the scales” or injected with formalin. The “young individuals and the adults of small species” were to be preserved in ethanol at 70° or formalin at 10°, noting that “a mix of formalin with methyl alcohol also works well, but the preservative solution needs to be changed after some time”. For tails of larger snakes, as in the case of the “surucucu [= puffadder, Bitis arietans], the authors mention that “caudal portions (…) of large specimens, always really fat, are difficult to preserve, needing special care” noting that it is “preferable to extract the skin and conserve it in alcohol”. In the case of amphibians, the authors noted that “alcohol at 70° preserves them well, but previous care of cleaning the skin mucous and injecting alcohol 90° in the abdomen” and that “the preservative fluid needs to be renewed, as, in these animals, it considerably drops the concentration”. For fishes “formalin at 8% or alcohol at 70° were used, taking with them the same care as with amphibians”. For invertebrates, Frade et al. (1946) reproduced Mendes (1942) table. Insect larvae and insects with “thick abdomens” were to be preserved in alcohol at 70°. No fluid preservation strategies were presented for birds. In a subsequent publication, the report on the zoological mission to Mozambique, Frade (1950) provided a brief overview of the collecting, preparation and preservation techniques used. These draw almost entirely, as the author noted, from those already used in Guinea-Bissau and published earlier (Frade et al. 1946). The only specific mention of fluid preservation that was somehow different than those already presented was the mention of scale insects and “soft” insects, that should be immersed in alcohol.

Such zoological missions resulted in the collections of the former Centro de Zoologia de Lisboa (CZL; Fig. 17) of the Instituto de Investigação Científica Tropical (IICT) (both now extinct), which were integrated in MUHNAC in 2015, where part of the collections is still extant. Ceríaco et al. (2021a) provided the history and a taxonomic overview of the herpetological collections (Fig. 18).

In the second half of the 20^th century, Museu Bocage experienced a rebirth, especially due to a new generation of naturalists that greatly influenced its activity during the following decades. One of these naturalists was the marine biologist Luiz Saldanha (1937–1997). Saldanha’s impact on Portuguese marine biology was remarkable, but he was also a museum person (see Ré et al. (2001)). In 1959, when he was still a biology undergraduate student at the Faculty of Sciences of the University of Lisbon (which shared space with Museu Bocage), Saldanha published a complete overview of preparation and preservation techniques for marine animals, ranging from sponges to fishes (Saldanha 1959). Saldanha provided a bibliographic review citing previous Portuguese publications (e.g. Bocage (1862); Oliveira (1895); Mendes (1942)) and other European (mostly French) references, but it is also fair to assume that some of the methods, techniques and formulas presented were those in use in Museu Bocage at this time. Across 32 tables, each dedicated to a specific taxonomic group and divided into four sections (Anaesthesia and death; Fixation; Preservation; Observations), Saldanha provided several options available to naturalists (Table 2).

Additionally, Saldanha (1959) also presented a list of the formulas mentioned in different methods and techniques, which we list and translate below:

• Anaesthetiser – Magnesium sulphate at the proportion of 154 g per seawater litre
• Bouin’s picro-formal-acetic fluid

• Picric acid (concentrated aqueous solution) 75 parts
• Formol 25 parts
• Acetic acid 5 parts

• Emollient – for specimens that are hardened by formol and have the body deformed

• Glycerol 10 parts
• Alcohol 10 parts
• Water 30 parts
• Fungicide (Niacine) as needed.

• Neutralised/buffered formol – in a dark jar, add some quantity of powdered calcium carbonate to the formol, that precipitates around 1 or 2 cm in the bottom of the jar.
• Perenyi liquor

• Acetic acid at 10% 400 cc
• Chromic acid at 0.5% 300 cc
• 90% alcohol 300 cc

• Chromo-acetic mixture

• Water 200 g
• Chromic acid 1 g
• Acetic acid 70 g

• Solution of hydrochloride of cocaine

• Cocaine 1 g
• Seawater 25 g

• Solution to euthanise and preserve fish and other vertebrates (including eggs)

• Alcohol 50 parts
• Water 50 parts
• Formol 5 parts

As an important note, Saldanha (1959) mentions that, whenever referring to “formol”, he is referring to “commercial formol”, which is usually sold as a 38% dilution. In 1965 Saldanha would publish an abridged two-page version of the earlier instructions, presenting mostly the same methods and techniques for the different groups of animals (Saldanha 1965). In 1968, Museu Bocage’s entomologist José de Almeida Fernandes (1931–2019) and Saldanha published a new booklet on how to collect, prepare and conserve zoological specimens (Fernandes and Saldanha 1968). This book was part of a course on natural sciences promoted by the Portuguese Society of Natural Sciences.

For the insects that “due to their nature (soft body, fragility etc.)” needed to be conserved in fluid, the authors suggested that they should be placed with their respective tags in small tubes/vials filled with either “alcohol 70%” or “agua formolizada” [“100 parts of water to 2–3 of formol”]. These tubes/vials should be closed with cotton stoppers and placed upside down in a larger jar (Fig. 19).

For marine invertebrates, before delving into the different taxonomic groups, Fernandes and Saldanha (1968) provide some brief biological generalities that generally apply to all groups. The authors noted that, as the marine animals “are generally contractible”, they needed to be anaesthetised before fixation. Compounds such as Nembutal, magnesium chloride and menthol could be applied indiscriminatelyto most of the groups for anaesthetisation. For fixation, the authors claimed that “it is usually done with formol at 5 or 10%, preferably in seawater or with alcohol at 70° or 96°” while, for preservation, this would generally be done in “commercial formol at 5%, if possible neutralised/buffered” or in alcohol at 70%”, noting however that, this would vary amongst different zoological groups.

For sponges, the authors suggested the specimen to be both fixed and preserved in alcohol at 70°, noting that the fixation could also be done with alcohol at 96° or absolute. While larger specimens could be preserved dry, a small part of them should be detached and preserved in alcohol at 70%. For Cnidarians, Fernandes and Saldanha (1968) presented different methods for the hydroids, medusae sensu lato, madreoporians, actiniarians and polyps, in general. Hidrarians would be anaesthetised with either menthol or magnesium chloride and both fixed and preserved in 5% formol. Medusae sensu lato should be left to die by asphyxiation in seawater or anaesthetised with menthol and then fixed and preserved in 10% and 5% formol, respectively. Madreporarians would simply be preserved dry after being cleaned with bleach for 12 hours. Actiniarians should be anaesthetised with menthol (but the authors warned that anaesthesia in these animals was usually slow), fixed in 10% formol and preserved in 5% formol. Finally, polyps in general should be anaesthetised with magnesium chloride, fixed in 10% formol and preserved in 70% alcohol. Annelids would be anaesthetszed with magnesium chloride for a couple of hours and both fixed and preserved in 70% alcohol, while, for crustaceans, 70% alcohol would be both narcotisation, fixation and preservation fluid. Molluscs should be anaesthetised with menthol or Nembutal and fixed and preserved with 70% ethanol, while echinoderms would be killed with 96% alcohol or 10% formol and preserved in 70% alcohol. Tunicates would be anaesthetised in magnesium chloride and both fixed and preserved in 10% formol.

For vertebrates, the authors suggested that fish should be euthanised in 5% or 10% formol and preserved either in 5% formol or 70° alcohol or in one of the following three solutions:

“a. Alcohol 50 p

Water 50 p

Formol 3 to 5 p

(This solution is also good for preserving the eggs of several groups of marine invertebrates).

b. Isopropanol 2 p

Water 3 p

To try to preserve the colour of fishes and amphibians, the following method should be used:

Fix in 8 or 10% formol (preferably neutralised/buffered) for two to four hours (or even a day).

Wash in running water.

Preserve in:

Water 400 p

Alcohol 600 p

Salt 20 g (sodium chloride)”

For amphibians and reptiles, the authors suggested that they could be killed with ether or chloroform or even be submersed alive in 10% formalin. They should then be injected with 10% formalin and be preserved either in 5% formol, 70% alcohol or the above cited “solution a” used in fishes.

Besides the specific instructions for each taxonomic group, Fernandes and Saldanha (1968) also provided some formulas and overall hints for fluid preservation. Picking on some of the information already provided by Saldanha (1959), the authors presented some formulas of the solution they referred to throughout the text, some of them that had not been presented in the previous publication. These were the cases of the magnesium chloride, which the authors explain to be “a solution of magnesium chloride at 7% in distilled water”, the menthol, whose crystals should be dispersed in the water where the anaesthesia was to be done and the Nembutal, which should be prepared as a solution with 3 mg per ml of sea water. For neutralising/buffering the formol, besides the method already presented in Saldanha (1959), the authors suggest that this could also be done with the use of hexamine (200 mg of hexamine per 1l of commercial formol). The authors also made some remarks on labelling and tagging specimens, noting that each specimen should have an associated label made of vegetable paper and written in China-ink or pencil inside the jar and that an external label glued outside the jar was also an option. The authors also refer that the collection should be kept in the dark.

Despite its almost anecdotal nature, it is also important to note the small paper about amphipods published in 1858 by Amílcar Mateus (dates of birth and death unknown, naturalist of the Instituto de Zoologia “Dr. Augusto Nobre” in Porto, where the author noted that these small animals could be simply preserved in a “flask of 70° alcohol or even aguardente” (Mateus 1958).

A few years later, Saldanha (1972) published a new and updated version of his 1959 paper on marine animals (Saldanha 1959). This was published in the journal “Arquivos do Museu Bocage”, the official publication of the Lisbon Museum. At this time, Saldanha was working as a researcher with a grant at Museu Bocage. Overall, this new paper followed the same structure and presented the same methods as those presented in Saldanha (1959), but also Fernandes and Saldanha (1968). It was, similar to the 1959 paper, organised as tables per taxonomic groups and had a formulae list at the end. The main differences between these two versions refer to specificities on how to anaesthetise, fix and preserve different types of animals, but also on the formulae list, which presented some additional formulae that were not present in Saldanha (1959) [although some of them were already presented in Fernandes and Saldanha (1968)]. These differences are presented in Table 3. In the formulae list, the main difference between the 1959 and 1972 versions was that the latter included the formulae/methods for applying magnesium chloride, menthol, Nembutal and sublimate (which were lacking in the 1959 versio,n but were already present in Fernandes and Saldanha (1968)), provided the option to neutralise/buffer the formol with hexamine (as in Fernandes and Saldanha (1968)) and lacked the reference to the solution of cocaine.

The specimens prepared by Saldanha and Fernandes were mostly lost in the fire that destroyed the Museu Bocage in 1978 (Fig. 20; Saldanha (1978)). The fire destroyed most of the zoological collections and the few fluid-preserved specimens that escaped this fire were sparred because they were on loan or due to simple luck, as the case of “154 specimens of common Portuguese and tropical species [of fishes]” that were “in one of the glass cases of the gallery of the fishes despite the adjacent destruction” (Saldanha 1978). In 1994, some of the specimens prepared by Saldanha and his team after the fire in the Laboratório Marítimo da Guia, a Marine Biology Laboratory associated with the Museu Bocage, were transferred to MUHNAC (Almeida and Pereira 2007), where they are still deposited.

In 1970, the naturalist Maria Helena Galhano (dates of birth and death unknown), associated to the Instituto de Zoologia “Dr. Augusto Nobre”, provided some very brief notes on how to collect, anaesthetise and fixate the poorly-studied groups of copepods, isopods and Mystacocarida. According to her, the specimens should be anaesthetised using “C¹²Mg” and then fixed in “70% alcohol or Bouin fluid” (Galhano 1970).

To our knowledge, no other publication regarding fluid preservation of zoological specimens has been published in Portugal in the last decades of the 20^th century or in the first decades of the 21^st century, so Saldanha (1972) is here assumed to be the last published naturalist instruction in the country. So-called “grey literature”, as non-published reports or field instructions only distributed to students and/or participants of research projects, may certainly exist across universities and research institutes in the country, but these are not easily accessible due to its almost private nature. Oral transmissions from professors, supervisors and international colleagues are also a common practice, but difficult to access and record.

The authors have declared that no competing interests exist.

No ethical statement was reported.

LMPC was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) of the Ministério da Educação do Brasil, under the Programa Institucional de Internacionalização (#PRINT, 88887.695166/2022-00 and 88887.978207/2024-00).

Conceptualization: LMPC, MPM. Data curation: MPM, LMPC. Formal analysis: LMPC, MPM. Funding acquisition: LMPC. Investigation: LMPC, MPM. Methodology: MPM, LMPC. Writing – original draft: MPM, LMPC. Writing – review and editing: MPM, LMPC.

Luis M. P. Ceríaco https://orcid.org/0000-0002-0591-9978

Mariana P. Marques https://orcid.org/0000-0002-1712-2632

All of the data that support the findings of this study are available in the main text.