Histochemistry: historical development

and current use in pathology

MA Riva1, M Manzoni2, G Isimbaldi2, G Cesana1, F Pagni2
1Section of History of Medicine, Research Centre on Public Health, and 2Department of Pathology,

San Gerardo Hospital, University of Milano Bicocca, Monza, Italy

Accepted July 2, 2013

Abstract
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We describe the history of the histochemical stains that contributed most to the development of
modern pathology during the last two centuries. Histochemical stains are presented in a list, which
provides the essential information about year, country and main use of each to enable the reader
to follow the chronological and geographical history of histochemistry. In addition to the histori-
cal evaluation of histochemistry development, we investigate how many classical histochemical
stains survive in a modern laboratory of pathology and how often they are used for diagnostic
practice compared to immunohistochemical (IHC) techniques. A ratio of about one histochemical
reaction to 13 IHC reactions was tabulated. Finally, our data make it possible to define different
cultural approaches to the terminology of histochemical and IHC stains: the former were based
on eponyms, which link the stain with the name of its inventor, while the latter use a more imper-
For personal use only.

sonal biological terminology.

Key words: histochemistry, history of pathology, immunohistochemistry

In a modern laboratory, a pathologist commonly
handles no more than 20 histochemical stains; he/
she often prefers to use more modern immuno-
histochemical (IHC) techniques that have almost
replaced histochemical stains in diagnostic and
research practice.
Histochemical stains have been the basis of
modern pathology, and have supported the devel-
opment of this discipline from the second half
of the 19th century until the 1960s. New genera-
tions of pathologists, trained during the IHC era,
are unaware of the fundamental role played by
histochemistry in the progress of pathology. We
describe here the histochemical stains that contrib-
uted most to the development of modern pathology
during the last two centuries and we present them
in a list that may be useful to current students of
medicine and pathology. In addition, by analyzing
Correspondence: Fabio Pagni, Department of Pathology,
University Milano Bicocca San Gerardo Hospital, 20900 Monza,
Italy. Phone: 0039 039 2332559, fax 0039 039 2332548, e-mail:
fabio.pagni@unimib.it
© 2014 The Biological Stain Commission
Biotechnic & Histochemistry 2014, 89(2): 81–90.
where and when these stains were developed, it is
possible for the reader to follow the most important
steps in the history of histochemistry and to obtain
essential information about the principal uses of
these stains.
Our clarification of past practices should be
compared with the current use of HC techniques:
the advent of IHC has revolutionized the approach
of the modern pathologist for diagnosis of diseases.
We investigate here how many classical histochemi-
cal stains survive in a modern pathology laboratory
and how often they currently are used in diagnostic
practice.
Material and methods
The principal histochemical stains that have been
used through the years for surgical pathology were
identified by perusing classic handbooks of his-
tochemistry (Lillie 1954, Gurr 1962). All of the origi-
nal papers that first described each technique then
were identified, together with the year of publica-
tion, the affiliation of the authors and the primary
field of application of each histochemical stain. The

DOI:10.3109/10520295.2013.822559 81
 stains were grouped according to the country where
they were developed, regardless of the nationality of
the developer (e.g., George Papanicolaou was born
in Greece, but his stain was classified as belonging
to the USA group). We also indicated the major field
of application for each stain. We focused especially
on hematoxylin because of its important role in
many different histochemical stains. All these data
subsequently were analyzed to evaluate the number
of eponyms that have been used for each dye.
Finally, we accessed the information system of
the Department of Pathology of San Gerardo Hos-
pital, University of Milano Bicocca, Monza, Italy, to
obtain a list of the histochemical stains used during
the period from November 1, 2011 to October 31,
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2012. Subsequently, we made a quantitative com-
parison between uses of IHC and traditional his-
tochemical staining.
Results
We selected 69 histochemical stains that are sum-
marized in Table 1 in chronological order of dis-
covery from 1858 to 2011. Owing to its peculiarity
and to its importance in the history of pathology,
For personal use only.
we researched hematoxylin separately (Table 2)
and identified another 11 variants; we gave a brief
description of each. We then tabulated all 80 stains
by year of discovery, first description and country.
In 58 of 80 cases (73%), the name of the inventor has
become the most common name for the stain.
Figure 1 lists the number of dyes discovered in
each decade from the 1850s to the 2010s; there are
two peaks, one between 1890 and 1930, and the other
between 1950 and 1960. The first peak accounts for
52% and the second for 25% of the histochemical
stains we studied. Figure 2 reports the country in
which each stain was developed; the USA and Ger-
many were the most productive countries in this
regard: 31 stains (39%) came from the US, 22 (27%)
from Germany, 8 (10%) from Italy and 5 (6%) from the
UK, while the remaining stains (18%) were developed
in another eight countries (Canada, Sweden, France,
Spain, Hungary, Nigeria, Russia, Czech Republic).
Initially, Germany and other European countries
developed the greatest number of stains, but they
were overtaken by the USA during the 20th century.
Figure 3 shows the major fields of applica-
tion using a radar chart that shows a prevalence
of general (owing to primarily to hematoxylin)
and connective tissue stains. Neuropathology and
hematopathology were the most productive disci-
plines for the development of new stains.
Finally, by analyzing the data from our labora-
tory for a one year period (November 1, 2011 to
82 Biotechnic & Histochemistry 2014, 89(2): 81–90
October 31, 2012) we found that the total number
of histological cases per year was 23,450. Figure 4
shows the use of histochemical stains during this
period; data are plotted on a logarithmic scale. The
total number of histochemical applications was
126,431, while during the same period, IHC reac-
tions numbered 20,954. Excluding hematoxylin-eo-
sin and Papanicolau staining that account for about
90,000 and 30,000 reactions, respectively, and are
used mainly for general tissue staining and gyne-
cological smears, we found a histochemistry:IHC
ratio of approximately 1:3. If we exclude the
routine Giemsa stain, mainly performed to detect
Helicobacter pylori in chronic gastritis specimens, the
ratio was 1:3 histochemistry:IHC.
Discussion
Histochemical staining techniques were first used
for scientific purposes in the second part of the
19th century and were adapted from methods in
use in the textile dyeing industry (Cook 1997). At
that time, commercial dye production was aimed
entirely at this industry and dyes were neither clas-
sified nor standardized with the needs of pathol-
ogy in mind. The first attempt to guarantee the
constancy and reliability of stains was made by the
German company, Grübler Co., but the real estab-
lishment of a biological dye supply industry began
with the creation in the USA of the “Commission on
Standardization of Biological Stains” in 1922 (Conn
1925), currently known as the Biological Stain
Commission.
Standardized dyes played a fundamental role
in the development of modern surgical pathol-
ogy by helping to confirm two primary theories
of the pathogenesis of diseases: the “theory of
membranes” postulated by Marie François Xavier
Bichat (1771-1802) and the “cellular pathology”
of Rudolf Virchow (1821-1902) (Zampieri 2012).
According to these theories, diseases are derived
from alterations of tissues and cells, respectively.
The application of these theories made it possible
to acquire a general knowledge of the histological
and cytological features of normal and pathologi-
cal tissues and cells. New tinctorial methods were
implemented, progressively moving from mono-
chromatic techniques, especially hematoxylin, to
two-dye sequential staining, e.g., hematoxylin-
eosin, then to the so-called trichrome techniques,
of which the first was Mallory’s technique in 1900
that was designed to demonstrate a wide range
of tissue constituents in contrasting colors (Titford
2009). The idea of selectivity of certain chemical
compounds for certain tissue components was
 Table 1. Introduction of new histochemical stains listed chronologically; country of origin plus main field of application are
noted. Stains based on hematoxylin are listed separately in Table 2

Stains Year Country Main field for use

Carmine stain (von Gerlach 1858) 1858 Germany general stain

Alizarin (Lieberkuhn 1864) 1864 Germany calcium
Perl’s Prussian blue (Perls 1867) 1867 Germany iron
Golgi (Golgi 1873) 1873 Italy neuropathology
Eosin (Fisher 1875) 1875 Germany general stain
Hematoxylin-eosin (Wissowzky 1876) 1876 Germany general stain
Ziehl-Neelsen (Ziehl 1882, Neelsen 1883) 1882 Germany microorganisms
van Gieson (van Gieson 1889) 1889 US connective tissues
Jenner (Jenner 1889) 1889 UK hematopathology
Romanowsky (Romanovsky 1891) 1891 Russia hematopathology
Nissl (Nissl 1894) 1894 Germany neuropathology
Mucinhematein (Mayer 1896) 1896 Italy mucopolysaccharides
Mucicarmine (Mayer 1896) 1896 Italy mucopolysaccharides
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Sudan III (Daddi 1896) 1896 Italy connective tissues

Weigert (Weigert 1898) 1898 Germany connective tissues
Mallory’s trichrome stain (Mallory 1900) 1900 US connective tissues
Leishmann (Leishmann 1901) 1901 UK hematopathology
Sudan IV (Michaelis 1901) 1901 Germany lipids
von Kossa (von Kossa 1901) 1901 Hungary calcium
Giemsa (Giemsa 1902) 1902 Germany hematopathology
Wright (Wright 1902) 1902 US hematopathology
May-Grunwald (May and Grunwald 1902) 1902 Germany hematopathology
Bielschowsky (Bielschowsky 1902) 1902 Germany neuropathology
Pappenheim (Pappenheim 1908) 1908 Germany hematopathology
For personal use only.

Masson-Fontana (Fontana 1912) 1912 Italy neuropathology

Cajal (Cajal 1913) 1913 Spain neuropathology
Jones (Jones 1913) 1913 US connective tissues
Feulgen (Feulgen 1914) 1914 Germany nucleic acid
Azan-Mallory (Heidenhain 1915) 1915 Germany connective tissues
Kinyoun’s fuchsine (Kinyoun 1915) 1915 US microorganisms
Rio-Hortega (Rio-Hortega 1917) 1917 Spain neuropathology
Fouchet (Fouchet 1917) 1917 France bile
Whartin-Starry (Warthin and Chronister 1920) 1920 US microorganisms
Holzer (Holzer 1921) 1921 Germany neuropathology
Congo red (Bennhold 1922) 1922 Germany amyloid
Oil red O (French 1926) 1926 US lipids
Papanicolau (Papanicolau 1928) 1928 US general stain
Weil (Weil 1928) 1928 US neuropathology
Schmorl (Schmorl 1928) 1928 Germany myopathology
Masson’s trichrome stain (Masson 1929) 1929 Canada connective tissues
Brown-Brenn’s Gram stain (Brown and Brenn 1931) 1931 US microorganisms
Sudan Black (Lison 1934) 1934 France lipids
Bodian (Bodian 1936) 1936 US neuropathology
Gordon-Sweets (Gordon and Sweets 1936) 1936 US connective tissues
Gomori (Gomori 1939) 1939 US connective tissues
PAS (Hotchkiss 1948) 1948 UK mucopolysaccharides
Orcein (Kurnick 1948) 1948 US connective tissues
Kurnick (Kurnick 1950) 1950 US nucleic acid
Alcian blue (Steedman 1950) 1950 UK mucopolysaccharides
Kluver-Barrera (Kluver and Barrera 1953) 1953 US neuropathology
Naphthol AS-D (Gomori 1953) 1953 US myopathology
Movat’s Pentachrome stain (Movat 1955) 1955 Canada connective tissues
Grocott (Grocott 1955) 1955 US microorganisms
ATPase (Padykula and Herman 1955) 1955 US myopathology
Luxol fast blue (Green 1956) 1956 US phase contrast microscopy
Thioflavine (Vassar and Culling 1959) 1959 Canada amyloid
(continued)

Histochemistry in pathology 83
 Table 1. (continued)

Stains Year Country Main field for use

Hillarp (Falck and Hillarp 1959) 1959 Sweden neurosecretory tumors

Hellerstrom-Hellman (Hellerstorm and Hellman 1960) 1960 Sweden neurosecretory tumors
Marinozzi (Marinozzi 1961) 1961 Italy electron microscopy
Truant (Truant et al. 1962) 1962 US microorganisms
Alcian blue-PAS (Mowry 1963) 1963 US mucopolysaccharides
Sirius red (Sweat et al. 1964) 1964 US connective tissues
Sevier-Munger (Sevier and Munger 1965) 1965 US neuropathology
Spicer’s high iron diamine (Spicer 1965) 1965 US mucopolysaccharides
Succinate dehydrogenase (Lojda 1965) 1965 Czech Republic myopathology
Grimelius (Grimelius 1968) 1968 Sweden neurosecretory tumors
Gurr (Gurr 1969) 1969 UK hematopathology
Brown-Hoops’s Gram stain (Brown and Hoops 1973) 1973 US microorganisms
Goldner’s trichrome stain (Goldner 1983) 1983 US connective tissues
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Table 2. Introduction of new hematoxylin stains listed in chronological order; place of introduction, the mordanting metal
ion and comments on use are given also

Hematoxylin Year Country Mordant Use as “general tissue stain”

Bohmer (Bohmer 1865) 1865 Germany Aluminum The first hematoxylin described
Heidenhain (Heidanhain 1885) 1885 Germany Iron Cytological stain for thin sections, to achieve
better detail. It stains tissues black and gray,
For personal use only.

which makes it useful for photomicrography.

It can be counterstained with acid stains such
as van Gieson.
Ehrlich (Ehrlich 1886) 1886 Germany Aluminum Counterstained with eosin, it is used to
demonstrate general tissue structures. The
glycerin content stabilizes the stain and
prevents over-oxidation; acetic acid results in
better nuclear staining.
Mayer (Mayer 1891) 1891 Italy Aluminum More vigorous than Ehrlich’s stain; when used
progressively shows better nuclear detail. It
gives little or no staining of
mucopolysaccharides
Harris (Harris 1900) 1900 US Aluminum A strong nuclear stain routinely used in exfoliative
cytology and for staining sex chromosomes.
Weigert (Weigert 1904) 1904 Germany Iron The standard iron hematoxylin used in the
laboratory, especially for demonstrating cell
nuclei (combined with van Gieson and
trichromic) of muscle fibers and connective
tissues. The resulting color is purplish red.
Verhoeff (Verhoeff 1908) 1908 US Iron Used for staining elastic tissues.
Carazzi (Carazzi 1911) 1911 Italy Aluminum A pale and precise nuclear stain, does not stain
cytoplasmic content. Suitable for frozen
sections.
Cole (Cole 1943) 1943 US Aluminum Routine stain for tissues, an alternative to Erlich’s
stain; it may be used in place of Mayer’s
hemalum in the celestin blue stain.
Gill (Gill et al. 1974) 1974 US Aluminum This hematoxylin neither overstains nor requires
differentiation.
Iyiola-Avwioro (Iyola and 2011 Nigeria Aluminum The stain keeps well; a major advantage of the
Avwioro 2011) new hematoxylin is that it does not form a scum
when stored, which makes filtration
unnecessary before use.

84 Biotechnic & Histochemistry 2014, 89(2): 81–90

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Fig. 1. Time course of introductions of new histochemical stains as listed in Tables 1 and 2. Numbers per decade are
noted on the chart.

the basis of not only modern pathology, but also this field and a Nobel Prize winner in medicine
modern pharmacology and chemotherapy. For in 1908, developed the use of some dyes in this
example, Paul Ehrlich (1854-1915), a pioneer in direction; he used methylene blue and trypan red
For personal use only.

Fig. 2. Illustration of geographical locations in which new histochemical stains as listed in Tables 1 and 2 were devised
and first used.

Histochemistry in pathology 85
Biotech Histochem Downloaded from informahealthcare.com by Nyu Medical Center on 10/12/14
Fig. 3. Major fields of application of the 80 stains listed in Tables 1 and 2, presented as a radar chart.
to treat malaria and trypanosomiasis, respectively
(Coleman 2006).
The period between the 1880s and 1930s marked
the birth and early development of histochemistry.
Geographically, development occurred primarily
For personal use only.
in Germany. In the USA, microscopists initially
employed natural dyes such as carmine, indigo,
alizarin and hematoxylin, but they also used the
first synthetic pigments such as eosin and Prussian
blue (Coleman 2006). These early histochemical
techniques were directed mainly toward identifying
single components of tissues: iron (Perls 1867), lip-
ids with Sudan III (Daddi 1896), mucus with muci-
carmine (Mayer 1896), calcium (von Kossa 1901),
mycobacteria (Ziehl 1882, Neelsen 1883), spirocheta
(Whartin and Chronister 1920) and Gram-positive
bacteria with Brown-Brenn’s stain (Brown and Brenn
1931). Considering the prevalence of infectious dis-
eases during that period, it is worth recalling
Fig. 4. Use of histochemical stains from November 1, 2011 to October 31, 2012 in the Dept. of Pathology, San Gerado
Hospital, Monza, Italy.
86 Biotechnic & Histochemistry 2014, 89(2): 81–90
that hematological stains (Romanowsky and its
variants, Giemsa, Wright and May-Grunwald)
were developed initially for studying infectious
diseases, particularly for detecting malaria infected
blood cells (Barcia 2007, Krafts and Pambuccian
2011, Dolan 2011, Dunning and Safo 2011). More
specific methods were implemented mainly in the
field of neuropathology (Nissl 1894, Bielschowsky
1902, Rio-Hortega 1917, Holzer 1921, Weil 1928)
for studying connective tissue with Azan-Mallory
(Heidenhain 1915) and trichrome staining tech-
niques (Masson 1929), and for identifying specific
components of tissues, e.g., lipids with oil red O
(French 1926), nucleic acids (Feulgen 1914), amy-
loid with Congo red (Bennhold 1922), melanin with
Masson-Fontana (Fontana 1912) and bile (Fouchet
1917). The first description of the Papanicolau
method (Papanicolau 1928), the basis of modern
cytopathology, also belongs to this period.
 With regard to the development of neuropathol-
ogy, it is worth recalling two giants, Camillo Golgi
(1843-1926) and Santiago Ramón y Cajal (1852-1934),
whose discoveries marked the beginning of modern
neurosciences (Schoenberg and Schoenberg 1979).
Our conception of neurons and neural networks
comes from their work; they made possible in depth
probing into the cerebral matter, a truly new discov-
ery and conception of the brain that was founded on
a beautifully stained network of neuronal nodes.
Also during these decades, histochemical stains
enabled identification of the etiological agents of
infectious diseases, thus confirming the “germ the-
ory” of Louis Pasteur (1822-1895) and the postulates
of Robert Koch (1843-1910) (Rosen 1993).
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From the 1940s onward, relatively few significant
histochemical staining techniques emerged. Some
fundamental stains, however, such as the trichrome
method (Gomori 1939) for endocrine granules and
the periodic acid-Schiff (PAS) technique (Hotchkiss
1948) for mucus, were developed during the period.
The definition of the classic globules of mucus in the
“signet-ring cells” of gastric carcinoma produced by
PAS staining, which has become an essential diag-
nostic tool for this tumor, is still used today.
For personal use only.
The second peak period produced some clas-
sic histochemical stains for myelin (Kluver and
Barrera 1953), mycetes (Grocott 1955), mucin with
Alcian blue PAS stain (Mowry 1963), neurosecretory
tumors (Grimelius 1968), as well as the majority
of myopathology histo-enzymatic stains includ-
ing naphthol ASD acetate esterase (Gomori 1953),
ATPase (Padykula and Herman 1965) and succinate
dehydrogenase (Lojda 1965).
During the 1980s, the use of histochemical stains
began to decline owing to the revolutionary devel-
opment of IHC. Only a few histochemical stains
still are used; the easy to use stains are especially
favored. Most of the special histochemical stains
that had mainly a descriptive goal or required dif-
ficult interpretations were largely abandoned. By
our analysis, 13 IHC stains are used for every one
histochemical stain. Generalizing from the results
of the activity in our department (curiously, the
founder of our hospital, St. Gerard, was a member
of a 12th century wealthy family of dyers) (Riva
and Cesana 2012), we can suggest that a few dyes
(hematoxylin-eosin, Papanicolau, and Giemsa)
still are widely used because of their role as gen-
eral staining methods. Reticulin, Perls, azan, PAS/
PAS-D, and Ziehl-Neelsen survive in pathology
departments for evaluating bone marrow fibrosis,
iron deposits, liver biopsy fibrosis, mucopolysac-
charides and mycobacteria, respectively. Others
(Grocott, Orcein, mucicarmine, Congo red) are
quantitatively under-used, but maintain high value
because of their specificity. Finally, a large group
of stains, the most important of which are Masson,
Weigert, Alcian blue, thioflavine and Kluver-Bar-
rera, are used sporadically, so we classified them as
declining dyes.
In view of this historical perspective, our analysis
shows that most histochemical stains derived their
names from the inventors instead of their chemical
composition. By contrast, IHC and molecular biol-
ogy techniques developed a technical nomenclature
based mainly on the antigens detected. In the new
molecular era, characterized by a multidisciplinary
approach to medical research, a single investigator
is not likely to give his or her name to a new stain-
ing technique. This transition occurred during the
second part of the last century, when there was a
shift from a morphologically based approach to a
functionally based approach.
The invention of IHC techniques belongs to the
early forties when Coons first described the “immu-
nological properties of antibodies containing a fluo-
rescent group” (Coons et al. 1941). In more recent
years, not only IHC techniques, but also the grow-
ing involvement of molecular biology in diagnosis
has accelerated the decline of histochemical stains.
IHC techniques therefore provide what we may
define as a functional stain that enables quantifica-
tion of cell activity that was impossible before its
development.
Before the introduction of IHC, a pathologist
was considered a morphologist of tissues and
cells, and his/her devotion to this field reached its
height in the search for a more perfect hematoxylin
stain (Coons et al. 1941, Allison 1999, Titford 2005,
Avwioro 2011). This stain became so important
for surgical pathology that it maintains the role of
“queen of stains” also during the IHC era. We identi-
fied 11 formulations, most of which originated from
US, German and Italian investigators, but interest in
this dye has never disappeared; the latest contribu-
tion came from two Nigerian authors (Iyiola and
Avwioro 2011).
Conflict of interest statement. The authors declare
no conflict of interest with regard to the contents of
this paper.
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