Histochemistry and Cell Biology (2024) 162:3–12

[Link]

REVIEW

A brief history of the Feulgen reaction

Marco Biggiogera1 · Margherita Cavallo1 · Claudio Casali1

Accepted: 19 March 2024 / Published online: 12 April 2024

© The Author(s) 2024

Abstract
One hundred years ago, Robert Feulgen published a landmark paper in which he described the first method to stain DNA in
cells and tissues. Although a century has passed since the discovery by Feulgen and Rossenbeck, the chemical reaction still
exerts an important influence in current histochemical studies. Its contribution in diverse fields, spanning from biomedicine
to plant biology, has paved the way for the most significant studies that constitute our current knowledge. The possibility
to specifically explore the DNA in cell nuclei while quantifying its content makes it a contemporary and timeless method.
Indeed, many histocytochemical studies following the 1924 paper have led to a deep understanding of genome organization
in general as well as several specific mechanisms (e.g. DNA duplication or tumour pathology) that, nowadays, constitute
some of the most fundamental pillars in biological investigations. In this review, we discuss the chemistry and application
of the Feulgen reaction to both light and electron microscopy.

Keywords Feulgen reaction · Schiff-type reagent · Osmium ammine · Light microscopy · Electron microscopy

Introduction term originally employed by histologists to refer to anything

within the nucleus that could be stained or coloured (chro-
The year 1924 marked a significant milestone in the mos in Greek, meaning colour), yet it lacked precise deline-
advancement of histocytochemical studies. That was the ation of the underlying chemical properties. The necessity
year when Feulgen and Rossenbeck published a chemical to provide clear answers to these questions led them to the
reaction which allowed the specific staining of deoxyri- development of a chemical reaction which allowed nucleic
bonucleic acid (DNA) in histological specimens (Feulgen acids to be specifically stained within the nucleus. A piv-
and Rossenbeck 1924). At that time, Watson and Crick’s otal role was played by Kossel and Steudel who initially
studies on the structure of the DNA double helix were still characterized the nitrogenous composition of what was then
far in the future and the scientific community had divergent referred to as thymonucleic acid owing to its thymine con-
opinions regarding the presence of DNA within cell nuclei. tent. It was observed that after its cleavage, for instance with
Although acknowledging the studies of Miescher who had nitric acid, it was capable of reducing Fehling’s solution
demonstrated the presence of a phosphorous-rich acid pre- thus resulting in the splitting of purine bases (Jones and
sumably present in the nuclei of purulent cells (Miescher Austrian 1907; Leven 1922; Jones 1953). Taking this infor-
1871), Feulgen and Rossenbeck themselves raised questions mation into consideration, Feulgen and Rossenbeck devised
about the actual nuclear presence of nucleic acids, recogniz- a method involving a first acid hydrolysis step that allowed
ing the difficulties arising when isolating them from complex the separation of purine bases thereby exposing free alde-
tissues since there was no guarantee that the extracted mate- hyde groups in the DNA structure. These aldehydes could
rial actually came from nuclei. Central to their inquiry was then selectively react with the Schiff reagent, also known
the concept behind the meaning of chromatin, which was a as basic fuchsin, which confers a purple-like coloration to
the sample. The peculiarity of this reaction is the fact that it
specifically stained the DNA as they observed that only thy-
* Marco Biggiogera monucleic acid-containing samples gave rise to a coloured
[Link]@[Link] product (Feulgen and Rossenbeck 1924).
1
Cell Biology and Neurobiology Laboratory, Department
It is also important to mention the involvement of
of Biology and Biotechnology “Lazzaro Spallanzani”, Frieda Feulgen, who gave an impactful contribution to the
University of Pavia, Via [Link] 9, 27100 Pavia, Italy

Vol.:(0123456789)
4 Histochemistry and Cell Biology (2024) 162:3–12

understanding of this chemical reaction; indeed she dis- by comparing a wide range of temperatures (between 9 and
cussed her doctoral dissertation, entitled “Untersuchungen 75 °C) and acid concentrations (from 6 to 0.05 M). From
über die Nuklealfärbung” (Investigations of Nuclear Stain- these findings, Kjellstrand concluded that the traditional pro-
ing), in 1924, the same year in which Robert Feulgen pub- cedure should be replaced with higher HCl concentrations
lished his work (Benedeum and Mesch 1999). and lower temperatures (e.g. 5 N HCl at room temperature)
Since then, the Feulgen reaction has significantly contrib- (Kjellstrand 1977).
uted to various fields spanning from plant biology to human An additional factor that might impact and interfere with
biomedicine elucidating numerous unanswered questions. acid hydrolysis is the type of fixative employed. Feulgen
Considering the influential scientific legacy that the Feulgen initially recommended the use of a sublimate-acetic fixa-
reaction brought with it, here we discuss the impact of this tive since he considered other fixatives containing oxidiz-
cytohistochemical method, highlighting the ongoing signifi- ing compounds potentially capable of damaging the nuclear
cance and utility of this technique which remains one of the material. In 1939 Hillary noted that the duration of hydroly-
most widely used in biology. sis depended on the type of fixative. He observed that chro-
mic acid did not interfere with the staining process; however,
a considerably longer period for acid hydrolysis was required
Chemical insights of the Feulgen reaction whereas fixation with acetic-ethanol required less (Hillary
1939). A decade later, Swift suggested fixation with for-
A key step in the Feulgen reaction is the detachment of maldehyde since he observed a higher yield compared to
purines from the deoxyribose sugar during the initial acid acetic-ethanol fixed specimens (Swift 1950). However, sub-
hydrolysis step (Fig. 1a), thus unmasking free aldehydic sequent groups found that acetic-ethanol fixatives yield the
groups on the DNA backbone which becomes apurinic. maximum intensity in plant materials (Sharma and Sharma
Exposed aldehydes specifically react with the Schiff rea- 1980).
gent whose main component consists of bleached para-
rosaniline that acquires a magenta colour upon DNA bind- Schiff stain
ing. Importantly, the Feulgen reaction is specific for DNA
staining since RNA is characterized by a ribose sugar, which The second phase of the Feulgen reaction consists of expos-
possesses an –OH group at the 2′ position, preventing acid ing the sample, whether fresh or fixed, to the Schiff reagent
hydrolysis and thus impeding purine detachment (Chieco (Fig. 1c). The latter, derived from basic fuchsin, is composed
and Derenzini 1999). of a pararosaniline dye, a member of the triaminotriphenyl-
methane dye family characterized by three phenyl groups
Acid hydrolysis attached to a central carbon atom (Delamater et al., 1950).
The Schiff reagent is prepared by bubbling ­SO2 through a
The original method proposed by Feulgen and Rossenbeck 0.5% solution of pararosaniline chloride until saturation,
(1924) consisted of a treatment with 1 N HCl at 60 °C for causing sulfur dioxide to bind to the central carbon atom or
4 min. However, the kinetics of acid hydrolysis depends on chromophoric structure, altering its structure and forming
multiple factors such as acid concentration, time and tem- a sulfonic acid compound, thereby bleaching the dye. Over
perature, making the initial step of this method crucial for time other compounds have been explored as a source of
achieving optimal yields. Indeed, as shown in Fig. 1b, as ­SO2, for instance potassium metabisulfite (­ Na2S2O5) (Kasten
hydrolysis progresses the value along the ascending branch 1960; Chieco and Derenzini 1999).
of the curve increases, suggesting a detachment of purine Upon binding with apurinic acid aldehydes, the reagent
bases from the DNA. Following a plateau stage, which indi- assumes its characteristic colour, staining the DNA magenta
cates the maximum level of DNA depurination, the curve (Fig. 2). However, numerous theories have emerged regard-
displays a descending trend indicating DNA breakdown and ing the mechanism of this reaction, and its chemical behav-
solubilization as reviewed by Mello and Vidal (2017). Con- iour remains a subject of ongoing debate. The most widely
sequently, several histocytochemical studies have focused accepted hypothesis, as discussed by Puchtler et al. in 1975,
on exploring different variables that could potentially influ- suggests that ­SO2 groups, upon reacting with aldehydes,
ence and optimize this initial phase. For instance, in 1972, form alkylsulfonic acid, wherein the carbon atom of the
Andersson and Kjellstrand demonstrated how, although both alkyl group binds to the nitrogen atom of the pararosaniline
acid concentration and temperature influenced the reaction primary aromatic amine, thereby restoring the chromophoric
speed, changes in HCl concentration corresponded to dis- function (Puchtler et al. 1975). These findings were consist-
tinct purine extraction rates (Andersson and Kjellstrand ent with those reported at the turn of the twentieth century
1972). Five years later, Kjellstrand evaluated the exposure by Prud’homme, who demonstrated that the reaction prod-
and removal of aldehydes during Feulgen acid hydrolysis ucts of basic fuchsin, sodium bisulfite and formaldehyde are
Histochemistry and Cell Biology (2024) 162:3–12 5

Fig. 1  a Schematic representa-

tion of a DNA nucleotide in an
acidic environment. Hydrogen
ions mediate the detachment
of guanine, therefore leaving
the DNA nucleotide apurinated
(readapted from Pourshahian
2021). b Typical Feulgen
hydrolysis curve. The x-axis
represents the time required for
hydrolysis and the y-axis indi-
cates the Feulgen–DNA values.
The plateau corresponds to the
maximum level of DNA depuri-
nation while the ascending and
descending branches repre-
sent DNA depurination and
depolymerisation, respectively
(modified from Mello and Vidal
2017). c One of the possible
interpretations of the chemical
reaction occurring when the
Schiff reagent interacts with
aldehyde groups. According to
this hypothesis, the aldehydic
group forms an alkylsulfonic
acid when interacting with S­ O2
groups; the binding of the C
atom from the alkyl group with
the primary aromatic amine
re-establishes the chromophoric
function of the dye, conse-
quently producing a magenta
staining (modified from Hubbe
et al. 2019)
6 Histochemistry and Cell Biology (2024) 162:3–12

confirming their results (Lessler 1948; Mirsky and Ris 1949;

Swift 1950). Therefore, following Mirsky and Ris’s work,
DNA content investigation became a widely used tool to
explore cell and nuclear mechanisms. For instance, Patau
and Swift demonstrated that cells double their DNA con-
tent prior to mitosis and provided insightful observations
concerning the changes occurring in chromosome structures
throughout mitosis, thus paving the way for all the studies
that have led to our current understanding of the cell cycle
(Patau and Swift 1953). DNA quantitative analyses through
colour measurements became widely employed in both
light microscopy and spectrophotometric analyses leading
to interesting investigations. For instance, Salisbury et al. in
1961 noted a decrease in the DNA colour density of bovine
spermatozoa upon aging; Cunningham and colleagues inves-
tigated the DNA content in nuclei of normal and neoplastic
rat tissues finding a significant increase in the second condi-
tion (Salisbury et al. 1961; Cunningham et al. 1950). Nota-
bly, these analyses had an impact on epigenetic studies and
significantly contributed to provide new insights into hetero-
chromatin and euchromatin features. In this regard, Mello
explored DNA organization in Malpighian tubes of Triatoma
infestans by observing spectral profiles of the specimens
which exhibited a shoulder at λ = 530 nm. The prominent
values were associated with heterochromatin areas due to
a higher amount of repetitive DNA. Higher signal intensity
Fig. 2  HeLa cells in mitosis after Feulgen reaction and observed in heterochromatin areas was due to the proximity of Schiff
under phase contrast. a Anaphase; b Telophase and late telophase. base molecules which disubstitute free aldehyde groups that
Courtesy of Carlo Pellicciari. Bar = 10 µm are close to each other. This approach paved the way for the
investigation of purine base proximity in chromatin regions
(Mello 1978).
alkylated and sulfonated derivatives of the parent compound
(Prud’homme, 1900).
More than historical remnants: a brief
selection of recent Feulgen reaction
DNA quantitative analysis applications

Despite the debate over the dynamics of the chemical reac- Despite being a now-distant milestone in the field of his-
tion, Feulgen’s method remained one of the principal means tochemical research and being a foundational method in
used to specifically detect and quantify DNA. Nowadays, it the field of nuclear cell biology, considering its poten-
is well established that the final colour density is propor- tial in nucleic acid staining and quantitation, the Feulgen
tional to the number of aldehyde groups on DNA (Kasten reaction is still being utilized for the modern investigation
1959; Dujindam and Van Duijn 1975). Initially, however, of multiple facets of DNA and, consequently, cell nuclei.
there was scepticism in this regard, e.g. Lessler questioned For instance, in a study of hepatocarcinogenesis, Taki-
the reliability of a DNA cytochemical quantification as moto and colleagues recently demonstrated the formation
hypothesized by Widström and Caspersson years before of Feulgen-positive cytoplasmic inclusions following
(Widström, 1928; Caspersson 1936). However, the criticism methylcarbamate exposure, indicative of potential chro-
did not stop the observations of Ris and Mirsky, who, in mosomal instability (Takimoto et al. 2024). In 2023, Alkan
1949, demonstrated that the DNA content is approximately and Koroglu-Aydin took advantage of the Feulgen stain of
constant within nuclei of one species (Mirsky and Ris 1949). oral epithelium cells to define the histopathological and
They evaluated the intensity of the reaction by absorption genotoxic effects of smoking and periodontitis, focus-
measurements with the optical microscope and compared the ing on DNA damage and micronuclei formation (Alkan
data obtained with those acquired with a spectrophotometer and Koroglu-Aydin 2023). Similarly, in 2022, Paiva and
Histochemistry and Cell Biology (2024) 162:3–12 7

colleagues combined the Feulgen reaction with other his- The quest for a Feulgen‑type reagent
tochemical stains in the study of oral mucosa exposed to for electron microscopy
carcinogens (Paiva et al. 2022). In addition, Kobayashi
et al. investigated nuclear profiles in lung carcinoma using The Italian and, foremostly, the Pavia School of His-
the Feulgen reaction, with particular attention given to tochemistry have been fertile ground for the usage and
the quantitation of the nuclear DNA content (Kobayashi advancement of the Feulgen reaction. A significant
et al. 2019). improvement was the development of novel, more sta-
These examples of recent applications clearly show the ble, and easily reproducible Schiff-type reagents, aiming
timelessness and impact of this foundational histochemi- to overcome limitations such as the intrinsic difficulties
cal technique. However, although the main field reported inherent in completing the laborious synthesis steps. These
here is associated with human biology, particularly can- innovations allowed the Feulgen reaction to efficiently
cer research, it is important to remark that the Feulgen expand its histochemical applications into areas such as
reaction has crossed barriers between different biologi- flow cytometry, where it served for quantitative DNA
cal areas. Concerning wider outlooks, Fidler and Gomes determination for both research and clinical purposes,
combined the qualitative and quantitative results of the and in particular electron microscopy (EM), granting DNA
Feulgen reaction for precise estimation of chromatin con- visualization with ultrastructural resolution (Casali et al.
densation and cellular rearrangements of the cerebellar 2022; Mazzini 2024).
layers in X-ray-exposed mice (Fidler and Gomes 2023). Interestingly, although the Feulgen reaction has been
Furthermore, other recent works not specifically focused known since 1924, it took almost 50 years before finding
on human biology have exploited the DNA specificity of a reagent capable of satisfying the requirements for its use
the Feulgen reaction. We could not even speculate whether in EM. This looks like a paradox, but the reasons were
Robert Feulgen would have imagined that his technique substantial. First a Feulgen-type reagent should have at
could have been applied to characterize and distinguish least one ammino group and an electrondense core to be
between two different drosophilids, Zaprionus indianus visible by EM. Schiff’s reagent, in fact, is only very weakly
and Zaprionus sepsoides, species which differ in the size electrondense (Gautier 1976). The obvious steps to take
of their testes and can be told apart from each other by the were to (a) increase artificially Schiff electrondensity or
spermatogenesis-applied Feulgen reaction (de Almeida (b) completely change the reagent type.
Rego et al. 2013). Focusing on other animal cell investi- As for the first approach, many attempts were made
gations, Feulgen staining was applied to evaluate several (Gautier 1976), but the most promising was proposed by
nuclear parameters, such as shape and size, DNA content, Moyne (1973) utilizing thallium ethylate. The procedure,
and chromatin compaction, in lymphocytes and epithelial however, was quite long, involving en bloc acetylation
cells of canine origin (Dos Santos et al. 2019). Similar to block hydroxyl groups, en bloc hydrolysis, and then
morphometric aspects were also investigated by Giuliano finally Schiff staining and thallium counterstaining on thin
and colleagues in llama sperm nuclei, with specific atten- sections. Moreover, thallium is a highly poisonous metal.
tion dedicated to chromatin distribution and measurement These factors, together with the fact that the specimens
of the haploid DNA content (Giuliano et al. 2018). prepared with this procedure were only suitable for DNA
Not only animal but also plant cell research has relied staining and nothing else (thus preventing different stain-
on the Feulgen reaction to perform specific DNA analysis. ings on adjacent sections), finally doomed the technique.
Indeed, in 2020, the application of Feulgen staining was As for alternative approaches to adapt the Feulgen reac-
described for imaging of ovaries and developing embryo tion to EM, a few attempts must be mentioned. The very
sacs in maize (Kalinowska et al. 2020). During the same first involved the reduction of silver atoms where aldehydes
year, Wojtczak applied the Feulgen reaction to study the were present. Already in 1924 Feulgen and Volt had shown
spermatid differentiation of the alga Chara vulgaris, high- that after a mild hydrolysis, silver ions could precipitate on
lighting important similarities with mammal spermiogenesis the newly generated aldehyde groups on DNA (Feulgen and
(Wojtczak 2020). Volt 1924). Breitschneider (1949) first and Peters (1966)
These examples show that the Feulgen reaction represents later stained whole sperm heads or thin sections for EM
a valid tool in various biological applications, ranging from analysis. Silver, unfortunately, can also precipitate where
animal to plant cell biology, even in the 2020s—a hundred SH-rich proteins are, for instance, or where other silver ions
years after its first description. Remarkably, the Feulgen are present. The reaction, hence, was definitely interesting
reaction even found relevance during the COVID-19 pan- but only preferential and not specific (Hayat 1993).
demic, when it was used to investigate nuclei of mucosa A completely different attempt to solve this important
samples collected from patients with COVID-19 (Sadik et al. problem was the NAMA-Ur procedure (Testillano et al.
2023).
8 Histochemistry and Cell Biology (2024) 162:3–12

1991). This method involved en bloc staining and a long character. The reagent was called osmium ammine complex
series of passages and resulted in a highly contrasted end (OA) (Cogliati and Gautier 1973). This paper was the first to
product. Although suffering from the same drawbacks as report a specific Feulgen-type reaction for EM. The results
thallium, it has been rediscovered for serial sectioning and were remarkable: specific, extremely fine-grained, high reso-
reconstruction of an entire cell nucleus stained for DNA lution, useful to stain DNA as well as in PAS-type reactions
using this approach has been reported (Roquette et al. (periodic acid–Schiff, for polysaccharides), perfect for thin
2009). sections.
The most difficult way (and, finally, the successful one) A minor problem was its low contrast; a major problem
was the search for a new electrondense Schiff-type reagent. was its long and costly synthesis with few well-defined steps
While working at the Centre de Microscopie Electron- and many steps linked to the change in colour of the solu-
ique (CME) of the University of Lausanne, I (M.B.) had tion. Briefly, the synthesis worked in about 40–50% of the
the opportunity to meet Alain Gautier and look through cases, and after a certain period it was impossible to repro-
his archives of notebooks. For several years he carefully duce it in Lausanne, but it worked in the Derenzini lab, in
recorded the numerous compounds he tested in order to Bologna. Olins and coworkers (1989) eventually described
find the right reagent, including Bismarck brown, hexam- a new type of synthesis of OA which was then called OA-B.
mineruthenium, hexachloroosmate etc. (Fig. 3). The great The synthesis was reproducible and the reagent became
majority were discarded, but finally the CME group was able commercially available. So far, however, the chemical for-
to synthesize an osmium-based polyammine with Schiff-type mula of neither OA nor OA-B has been clarified or provided.

Fig. 3  From Alain Gautier’s archives. The text reads: Metallic positive or negative, while osmium ammine results were positive
polyam(m)mines. Reagents tested for a Schiff-type or Luft-type speci- in all the different batches utilized. The exception is represented by
ficity. Diamino dinitrite palladium; methylamine tungstate; osmium Osmeth (osmium methenammine) and hexaammineosmium triiodide
ammine complexes. As shown, palladium and tungsten are weakly
Histochemistry and Cell Biology (2024) 162:3–12 9

Applications of osmium ammine

The first papers that applied the Feulgen reaction to EM

(Cogliati and Gautier 1973; Gautier et al. 1973) were
mostly dedicated to the demonstration of the Schiff-type
qualities of OA. Only later came the biological applica-
tions. One of the first to embrace the technique was Moyne
(Moyne et al. 1978; Moyne 1980), who analysed the virus-
induced nuclear inclusions, revealing viral DNA before the
appearance of other nuclear alterations.
Most of the interesting papers came from the group of
Derenzini. In several publications they showed the struc-
ture of chromatin in situ in the nucleus (Derenzini et al.
1982, 1983) at the level of the thinnest chromatin fibres.
In a couple of papers (Hernandez-Verdun et al. 1982;
Hernandez-Verdun and Derenzini 1983) they studied
the structure of chromatin inside the nucleolus, describ-
ing the presence of areas of DNA without histones, in
a non-nucleosomal conformation. The high resolution of
the technique allowed them to visualize individual nucle-
osomes, highlighting nucleosomal DNA surrounding an
electron-lucent histone core (Hernandez-Verdun and Der-
enzini 1983) (Fig. 4).
Detection of DNA within individual mitochondria as
well as in viruses was shown to be feasible (Liu et al.
1992; Puvion-Dutilleul et al. 1996).
Another important application was the visualization of
the S-phase distribution of DNA in parallel with immu-
nolabelling for BrdU which demonstrated the possibility
to utilize OA after other cytochemical procedures (el-Alfy
et al. 1995). Indeed, the technique can be carried out after
immunolabelling provided that the thin sections are left
to dry for some time. Acid hydrolysis does not displace
or remove colloidal gold particles, thus allowing precise
localization of both DNA and the target proteins (Biggi-
ogera et al. 2001). OA was also proven to work perfectly
on cryosections (Puvion and Bernhard 1975).
Interestingly, detection of DNA at the EM level showed
some strange patterns in particular types of chromatin. Fig. 4  a P815 mouse mastocytoma cell, immunolabelled for nucleo-
lin and stained for DNA with OA-B. HCl hydrolysis 45 min, OA-B
In mature sperm nuclei in mouse (Biggiogera 1986)
staining 60 min. Note the intense nucleolar labelling and the highly
and other species (Courtens et al. 1991, 1994), the cell contrasted DNA. cy: cytoplasm; nc: nucleolus. Bar = 500 nm. b P815
nucleus, homogeneously stained after exposure to uranyl cell, OA-B staining. At high magnification note the fibrillar centre
and lead, becomes stained in spots, as if the DNA was (asterisk) and thin filament of DNA emerging from the condensed
DNA region the region. chr: condensed chromatin at the periphery of
unevenly distributed. The reason for this behaviour lies
the nucleolus. Bar = 100 nm
in microheterogeneities of the protamine–protamine inter-
action (Boutinard Rouelle-Rossier and Biggiogera 1992)
rather than reflecting biological features. Furthermore,
chromosomes and their characteristics were also studied thin rim of DNA surrounding the prenucleolar bodies.
with interesting findings related to their structure (Liu The stained DNA then “colonized” the entire nucleolus.
et al. 1995). For example, developing mouse embryos Several reviews summarize in depth the wide spectrum
(Fakan and Odartchenko 1980) were shown to exhibit a of applications of OA in revealing chromatin architecture
(Biggiogera et al. 1996; Derenzini et al. 2014; Biggiogera
2024).
10 Histochemistry and Cell Biology (2024) 162:3–12

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