Historically the development of dyes and the insight that
biological tissues or structures have a selective affinity for
certain dyes, has had a very important effect upon medicine. This
applies not only to microscopy, but also to the development of
chemotherapy. The sulphones and sulphamides had their origin in the
synthetic dye industry. This industry came into being because the
alternatives were unsatisfactory. In order to find out how this
came about historically, we will discuss the ways in which dyes
were manufactured in early times. [We will not discuss pigments
which are used for artist's paint].
In olden days Tyre, the capital of Phoenicia, was famous for its
purple. Tyrian purple was obtained from the hypobranchial gland of
certain molluscs (Murex trunculus, M. brandaris and others). These hermaphrodite
creatures contained more dye during their male phase than during
their female phase. The enormous numbers of animals needed (10,000
shells for 1 gram of dye), meant that the substance was
inordinately expensive. The knowledge of how to obtain purple was
lost during the middle ages. Until the middle of the
19th century dyes for textiles were chiefly of plant
origin. A grey, beige or pale brown colour can be obtained from
many plants, but clear fast and deep colours are not so easy to
obtain. The most important European plants from which dyes were
obtained for textiles were woad, weld and madder. Deep blue was
obtained from woad, red from madder and yellow from weld, sweet
gale and saw-wort.
These plants belong to widely different botanical families: woad (Isatis tinctoria, fam. Cruciferae), madder (Rubia tinctorum, fam. Rubiaceae), weld (Reseda luteola, fam. Resedaceae), saw-wort (Serratula tinctoria, Compositae), dyer's greenweed (Genista tinctoria, leguminous plants), bog myrtle, or gale (Myrica gale, fam. Myricaceae). Various lichens (Roccellatinctoria or orchil lichen) were used as sources for dyes on a small scale. Litmus is obtained from the same organisms and is still used today as a pH indicator. Safflower (Carthamus tinctorius, fam. Compositae) is a thistle-like plant from which a reddish yellow dye and also an oil is obtained. This is also still used today. Turmeric or kurkuma, (Curcuma longa, fam. Zingiberaceae) came from the Far East. This plant is still used today to colour various culinary dishes.
Indigo holds a special place among dyes. The famous scientist Isaac Newton identified seven colours in the prismatic spectrum at the end of the 17th century. He added orange (between red and yellow) and indigo (between blue and violet) to the 5 colours of the rainbow already recognised at that time, which led to heated discussions. Even now, many people consider indigo to be a hue of blue. The word indigo refers to the blue colour, the plant Indigofera and the dye. The latter substance is also known as indigotin. Indigo was cultivated in antiquity, chiefly in India. The name of the colour indeed refers to that country. The dye indigo is obtainable from many species of plant. The plants which supply a high quality of indigo only grow in warm climates. Polygonum tinctorium (Polygonaceae, the knotgrass family) and Strobilanthes flaccidifolius (Acanthaceae, Acanthus family) are important sources in the Far East. Several Indigofera species also provide indigo. Each plant species within this genus has its own preference as regards climate. These plants were cultivated on a larger scale than others. Since they belong to the papilionaceaous plants, they do not exhaust the soil, but enrich it with nitrogen (unlike woad, for example). These plants bear root nodules in which symbiotic Rhizobium bacteria fix atmospheric nitrogen by means of the oxygen-sensitive enzyme nitrogenase.
Woad contains the same dye as indigo but in much lower
concentrations. Woad provided the dye which the Picts, a Celtic
tribe in Britain, used to decorate their bodies or as warpaint in
their conflicts with the Romans (Lat. "picti": painted ones, Celtic
"brith": paint). Residues of blue paint were even discovered on the
body of the "Lindow man", a 2000-year-old Celtic corpse which was
found in a peat bog in England in 1987. The dye in woad is present
as colourless glycosides (i.e. bound to a sugar), in the substances
called indican and isatan. The leaves and stalks must be fermented
in an alkaline environment (urine, bird droppings, wood ash, lime)
to release the active dye. The yellowish green dye produced must
then be oxidated to obtain the blue. The fermenting process leaves
behind a legendary stench. In 1601 the English Queen Elisabeth I
forbade all woad processing within 10 kilometres of her residences.
The plant gives good results with wool, but much less beautiful
colours with cotton or fibres such as flax or hemp. In order to
obtain a beautiful deep blue colour, indigo was needed.
In the middle ages indigo, which was very expensive, was
imported into Europe in small quantities. Until the end of the
middle ages, all trade between Europe and India was via the Levant.
When Vasco da Gama sailed around the Cape of Good Hope in 1498, he
demonstrated the possibility of a direct connection between India
and Europe. The tax monopoly which the Muslim Middle East had on
all trade (including that in indigo) between the West and India was
broken. The historical importance should not be underestimated. The
trade in exotic goods and spices from the various East India
Companies which was blossoming at that time included indigo as well
as pepper, cloves, nutmeg, cinnamon, printed cotton, silk, perfume,
tea, opium, lacquer work, ivory, crystal and camphor. The indigo
trade together with the pepper trade were among the most lucrative
activities. Indigo was very much in demand. The blue obtained from
woad coloured textiles quite weakly. A much more beautiful, deeper
and faster blue could be obtained using indigo. The importation of
indigo from India threatened the domestic woad production in a
number of countries, and the importation of indigo from the East
was curbed. Later, European companies set up indigo plantations on
a large scale in India and various other overseas regions. The
import ban was then abolished.
The roots of madder were widely used until the end of the
19th century. The genus name of this plant
("Rubia") refers to the red dye (Turkish red) which can be
obtained from it (Lat. "ruber" = red). The plant contains
alizarine. This substance provides a beautiful red, orange, pink,
brown or purplish black dye, depending on the mordant used. Many
materials such as cotton and wool do not naturally absorb dyes,
with the exception of indigo. Indigo binds via hydrogen bridges to
the fibres, unlike other dyes, which need to be fixed covalently.
Mordants act on the textile to facilitate later absorption of dyes.
These are substances such as oxalic acid, tannin, tartrate
(tartaric acid) or alum (KAl(SO)4, or more correctly,
since this is a double salt
Metal salts are also often used as mordants, such as aluminium,
iron, chrome or tin salts. After the discovery of chemical
alizarine synthesis, the cultivation of madder was discontinued.
Nowadays alizarine red is still sometimes used in histology to
detect calcium in tissue sections (e.g. skeletal studies).
Not only herbal plants but also some wood species are used to dye textiles. A beautiful brown is obtained from the wood of cachou (Acacia catechu, fam. Mimosaceae), similar to acajou. Fustic (Chloroflora tinctoria, related to the mulberry tree) gives yellow. The discovery of the New World in 1492 opened up many new possibilities. In 1494 the Treaty of Tordesillas was concluded between Castile and Portugal. This treaty determined the division of all regions in the newly discovered New World. All regions West of the meridian running 370 leagues West of the Cape Verde Islands, were for Spain, all regions East of this line were for Portugal. In 1500 Pedro Alvarez Cabral discovered the coast of Brazil and sent a ship from his fleet back to Portugal with the news of this discovery. Among the goods loaded on board was the very hard brazil wood or pernambuco. It was quickly discovered that a red dye could be obtained from this. Brazil actually received its name from this plant. In fact there are several plants which carry this popular name. They include Caesalpinia echinata and C. sappan (fam. Caesalpinia) and Haematoxylum brasiletto (fam. Fabaceae). The latter name refers to the red colour (Gr. "Haima" = blood, "xylon" = wood). Due to the deforestation that followed, the wood soon became scarce and expensive. Nowadays pernambuco is still used for the best quality violin bows. Logwood, the wood of Haematoxylon campechianum, was also valuable. The etheric extract from the wood produced a wonderful black dye after using chromium oxide as mordant. Haematoxylin is still often used today (together with eosin) in histological preparations.
Several millennia ago, before the dawn of the Iron Age, the Sumerian civilization already had a word for iron: anbar. This word combines the root an (sky) with bar (metal), so literally "sky-metal", referring to meteoritic iron which was occasionally found and used. About 5% of the meteorites found on earth are made from iron, fragments of asteroid cores that were molten early in the history of the Solar System. Such iron meteorites typically contain nickel upwards of 5%. This natural alloy is harder than pure iron. It was more valuable than silver. The syllable bar lives on in modern words such as brass (alloy of copper and zinc) and the Latin ferrum (bar -> ber -> fer), the Hebrew word barzel for iron and possibly as "bra" in the term brazil wood for tropical hardwood (ironwood). (Alternative etymologies also exist, e.g. bresil -> breze referring to glowing charcoal). Who would have guessed that there might be a (somewhat twisted) connection between a common histological stain and "sky-metal" in pre-Iron Age Sumeria?
|Dactylopius coccus, source of cochenilla. The scale insect is crushed in the palm of the hand. Copyright ITM|
|Dactylopius coccus, source of cochenilla. The scale insect is in the palm of the hand. Photo Dr Van den Enden, Peru. Copyright ITM|
Cochenille is harvested from scale insects (Dactylopius coccus) which live on Opuntia cactus (Prickly pear).|
When the Spaniards conquered the New World, they quickly noticed that some Indian clothing was an unusually beautiful red, a colour which was then unknown in Europe. This scarlet was obtained from cochineal, which contains carminic acid, an anthraquinone glycoside. This beautiful red dye was prepared beginning with certain dried female scale insects (Dactylopius coccus, Homoptera). The small male insects of this species have wings but no mouth parts. In the week that they live, they have to try and fertilise as many females as possible. The female creatures live on various cacti (Opuntia sp. and Nopalea coccinellifera, known as the Prickly Pear). The fertilised females secrete a white substance which forms a thick crust in which the immobile creature herself is incarcerated while she drinks plant juice. This substance protects her from many predators, and from drying out. Some predators such as the caterpillar Laetilia coccidivora succeed, however, in consuming the scale insects in order to use the carminic acid for their own defence. The scale insects were at first regarded as a kind of seed ("grana"). Only later did their insect nature become clear.
The insect came originally from Mexico and was later imported into other countries of Central and South America, as well as the Canary Islands. The insects were scraped from the plant together with their waxy protective covering, and boiled in a cask together with oxalic acid-containing plants (tejute, Miconia argentea), to obtain the red colour. Later citrus was also used in the preparation. Citrus fruits were imported by the Spaniards and are not native to Mexico. Similar but less brilliant dyes (carmine or crimson) were obtained from kermes, the harvested and dried Kermes vermiglio (vermiglio refers to vermilion) and Kermes ilicis, scale insects which live on the kermes oak (Quercus coccifera, fam. Fagaceae). This plant is a bush or small tree which is widely distributed around the Mediterranean region. [Kermes should not be confused with shellac, a lacquer obtained from Lacciferalacca or Tachardia sp, scale insects which live on some Ficus species in Southeast Asia]. In Central Europe, Polish cochineal was obtained in small amounts from scale insects (Porphyrophora polonica). Armenian cochineal was obtained from a related insect (Porphyrophora hamelii). Nowadays natural dyes are used in small amounts, for example to colour hair. These include henna (obtained from Lawsonia inermis, fam. Lythraceae or purple loosestrife, and from the roots of Alcanna tinctoria, fam. Boraginaceae). Some have also found a place as additives to colour foodstuffs, such as annatto (E160b, obtained form the seedcoat of Bixa orellana), indigotin (E132) and cochineal red (E120). Food additives sometimes cause allergy. Plant and animal dyes have a number of disadvantages as regards fastness and depth of colour as well as availability and price. Cochineal was just as expensive as gold. Saffron, which is obtained from a crocus (Crocus sativa) is the most expensive spice or dye there has ever been. Brazil wood was very scarce and could not possibly meet the demand. Alternatives were diligently sought and the use of natural dyes swiftly diminished when synthetic dyes were introduced.
It had been known since 1826 that if indigo were heated, colourless aniline was formed. [The Sanskrit for dark blue is "nila", from which the name "aniline" comes]. Around 1830 the German chemist Friedlieb Runge was studying the composition of coal tar. He isolated phenol, which would later be used by Lister as an antiseptic. Aniline (= aminobenzene or phenylamine) was also isolated from coal tar. In 1856 the 18-year-old Englishman William Henry Perkin (1838-1907), in attempting to synthesise quinine, discovered the first synthetic dye mauveine, from aniline and potassium dichromate. This discovery, and the idea of making synthetic dyes which it gave rise to, had enormous consequences: the birth of the modern chemicals industry. In 1865 August Kekulé published his pioneering theory concerning the structure of benzene, which was a major breakthrough. The idea of making synthetic indigo began to take shape. A few years later the German pioneer in chemistry, Adolf von Baeyer (Nobel prize 1905) succeeded in synthesising indigo. The famous Tyrian purple proved to be a brome derivative of indigo.
Von Baeyer worked together with Heinrich Caro, head of the
research department of the Badische Anilin Soda Fabrik
(BASF). In 1868 von Baeyer's apprentices Karl Graebe and Karl
Lieberman perfected the synthesis of alizarine, based on
anthracene. Steel production increased enormously in Europe during
this historic period. For this, large amounts of cokes were needed.
Cokes, coal gas (for lighting) and coal tar were obtained by
heating coal in an oxygen-free atmosphere. At that time coal tar
was a useless and dirty waste residue of the industrial production
of cokes. Due to the increasing steel production, Prussia was left
with a huge toxic waste problem. Aniline could be obtained in large
amounts from coal tar and also formed the basis for synthesising
indigo. Instead of waste, the coal tar now became a raw material.
It was discovered that fractionation of coal tar could supply many
different basic substances for the production of various dyes. The
need for alternatives to natural dyes, market forces and the new
scientific insights led to the swift development of the dye
industry. Countless chemical products were discovered. BASF at
first made enormous profits from the industrial production of dyes.
Due to the cheap synthetic indigo, woad cultivation and the British
trade in Indian indigo collapsed. One of the consequences was that
the Indian farmers now had to pay their taxes in cash and not in
indigo. This led to an increase in poverty, and to an uprising
supported by Mahatma Gandhi. Indirectly it meant the beginning of
the movement for an independent India and the end of the British
|Chemical formula of ANILINE. Copyright ITM|
|Chemical formula of INDIGO. Copyright ITM|
Many dyes which we still use today for microscopic preparations
were perfected then, e.g. eosin, auramine, aniline derivatives such
as fuchsin, necessary for Ziehl staining (see also Romanovsky dyes,
chapter on Malaria). What connection does this have with antibiotic
agents? It was known that many bacteria absorb dyes. The variety of
dyes which could be extracted from plants was too restricted to
exploit this observation. Paul Ehrlich, a student of the anatomist
Wilhelm Waldeyer, ascertained that the dye methylene blue had a
very weak antimalarial action. Gentian violet and mercurochrome
(coloured aniline derivatives) were also active as antiseptics. The
question was asked whether following absorption a dye could be
sufficiently toxic to kill bacteria while they were present in the
human body. Ehrlich stated that parasites and micro-organisms
contained chemoreceptors which were sufficiently different from the
analogous structures in humans that dyes might bind to them
differentially. If the dye was also toxic, it might be used
therapeutically. To test this hypothesis, the German dyeworks I.G.
Farben, which was associated with BASF, started a programme to test
the thousands of products in their collection for antibacterial
action, and these included azo-compounds (an azo group consists of
-N=N-). Azo compounds can be derived from aniline. Thus in 1932
Gerhard Domagk, head of the department of experimental pathology
and bacteriology, discovered that the red azo dye Prontosil®
could destroy streptococci in vivo. In vitro the product proved
inactive. The discovery that this aniline derivative was split at
the azo group into an active part (sulphanilamide) and an
inactive part, supplied the basis for the further development of
sulphones (general formula R-SO2-R'; e.g. dapsone =4,5'-sulphonyl-di-aniline) and the sulphonamides (general
formula R-SO2NH-R'). The idea that a molecule could kill
a bacterium in vivo without interfering with the host's physiology,
was conceptually an enormous step forwards. Domagk was awarded the
Nobel prize in 1939 (he was not able to receive it until 1947,
however, due to the conditions in Nazi Germany). Dapsone became
widely available and the rational chemotherapy of leprosy could
As well as sulphones and sulphonamides, other products were also
derived from aniline, such as phenacetine, procaine, indomethacin,
lidocaine, benzocaine and many others. A common side effect of
these products is methaemoglobinaemia, chiefly in people who are
heterozygote for methaemoglobin reductase deficiency (= cytochrome
b5 reductase). The most famous products responsible for
this condition are dapsone, phenacetine, nitroglycerine,
nitroprusside and in particular benzocaine. Nitrites and nitrates
(food preservation, contamination of ground water, amylnitrite) can
also trigger this condition. In methaemoglobinemia the iron atom in
haem is oxidised (Fe2+ to Fe3+) and can no
longer bind oxygen. This also causes a left shift of the Hb-oxygen
dissociation curve so that oxygen is released less easily from the
tissues, which exacerbates hypoxia. If the concentration of
methaemoglobin is 10%, there is pronounced cyanosis, without signs
of heart or lung abnormalities. The arterial blood is as brown as
chocolate at levels above 15% and does not become redder after
contact with atmospheric oxygen. At concentrations around 35% there
is dyspnoea, weakness and headache. From 40% tissue ischaemia
occurs. Concentrations above 80% are often fatal. Standard pulse
oximetry is not reliable (false high values in methaemoglobinaemia
and false low values in methylene blue therapy). Methylene blue (2
mg/kg) reverses the reaction and is the antidote in acute
situations. Improvement can be expected after 30'-60'. Remember
that methylene blue can trigger haemolysis in people with G6PD
deficiency. In serious situations, an exchange transfusion and
treatment with hyperbaric oxygen can be carried out.