The Feulgen reaction: from pink-magenta to rainbow fluorescent at the Maffo Vialli’s School of Histochemistry

Published: 22 February 2024
Abstract Views: 140
PDF: 128
Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.


For over a century, Palazzo Botta (Palace Botta) has housed the University of Pavia's Biomedical Institutes. Illustrious scientists have conducted research and taught at this Palace, making significant contributions to the advancement of natural, biological, and medical science. Among them, Camillo Golgi received the Nobel Prize for discovering the so-called "black reaction." Following Golgi, the Palace continued to be a hub for the development of methodologies and reactions aimed at detecting and quantifying biological components. Maffo Vialli (in the Golgi stream) was the first to establish a Histochemistry Research Group, which began in the naturalistic field and later expanded to the biomedical area. Among the many histochemical studies initiated in the Palace, the Feulgen reaction undoubtedly played a significant role. This reaction, developed R. Feulgen and H. Rossenbeck in 1924, had significant international implications: numerous researchers then contributed to define its fine chemical details, which remained the subject of study for years, resulting in a massive international scientific literature. The Pavia School of Histochemistry also contributed to the evolution and application of this method, which has become a true benchmark in quantitative histochemistry. Giovanni Prenna and the CNR Centre for Histochemistry made significant contributions, as they were already focused on fluorescence cytochemistry. The Pavia researchers made significant contributions to the development of methodology and, in particular, instrumentation; the evolution of the latter resulted in the emergence of flow cytometry and an ever-increasing family of fluorescent probes, which somewhat overshadowed the Feulgen reaction for DNA quantification. The advent of monoclonal antibodies then contributed to the final explosion of flow cytometry in clinical application, almost making young neophytes forget that its roots date back to Feulgen.



PlumX Metrics


Download data is not yet available.


Vialli M, Erspamer V. Contributo alla conoscenza istochimica delle cellule entero- cromaffini. Arch. Ital. Anat. Embriol. 1936;37:411.
Erspamer V, Asero B. Identification of enteramine, the specific hormone of the enterochromaffin cell system, as 5-hydroxytryptamine. Nature 1952;169:800. DOI: The Nobel Prize in Physiology or Medicine 1906. Available from:
Feulgen R, Rossenbeck H. [Mikroskopisch-chemischer Nachweis einer Nukleinsäure von Typus der Thymonukleinsäure und die darauf beruhende selektive Färbung von Zellkernen in mikroskopischen Präparaten].[Article in German]. Hoppe-Seylers Z. Physiol Chem 1924;135:203-48. DOI:
Kasten FH. Robert Feulgen and his histochemical reaction for DNA. Biotech Histochem 2003;78:45-9. DOI:
Chieco P, Derenzini M. The Feulgen reaction 75 years on. Histochem Cell Biol 1999;111:345-58. DOI:
Duijndam WAL, Van Duijn P. The influence of chromatin compactness on the stoichiometry of the Feulgen-Schiff procedure studied in model films. II. Investigations on films containing condensed or swollen chicken erythrocyte nuclei. J Histochem Cytochem 1975;23:891-900. DOI:
Gil JE, Jotz MM. Further observations on the chemistry of pararosaniline-Feulgen staining. Histochemistry 1976;46:147-60. DOI:
Kjellstrand PTT. Temperature and acid concentration in the search for optimum Feulgen hydrolysis conditions. J Histochem Cytochem 1977;25:129-34. DOI:
Böhm N, Sprenger E. Fluorescence cytophotometry:a valuable method for the quantitative determination of nuclear Feulgen-DNA. Histochemie 1968;16:100-18. DOI:
Vialli M. [Su una possibile spiegazione di alcune presunte anomalie alla legge della costanza quantitativa dell’acido desossiribonucleico nelle cellule].[Article in Italian]. Rend Ist Lomb Sc Lett 1954;87:29-36.
Vialli M. [Ricerche di istochimica quantitativa comparata sull’acido desossiribonucleico e sulle proteine].[Article in Italian]. Monit Zool Ital 1954;62:28-41.
Vialli M. [Volume et contenu en ADN par noyau].[Article in French]. Exp Cell Res 1957;13:S284-93.
Vialli M, Reggiani M. [Dispositivo per lo studio colorimetrico e fotometrico di preparati microscopici].[Article in Italian]. Boll Soc Med Chirur Pavia 1948;62:299-301.
Casella C, Reggiani M.[ Istospettrografia di fluorescenza].[Article in Italian]. Arch Biol 1949;60:207-34.
Vialli M, Romanini G. [Dispositivi semplificati di istofotometria nel visibile].[Article in Italian]. Boll Soc Ital Biol Sperim 1950;26:1633.
Vialli M, Zanotti L. [Due nuovi modelli di istofotometri per assunzione di curve di assorbimento].[Article in Italian]. La Ric Scient 1957;27:3.
Vialli M, Perugini S. [Due nuovi modelli di apparecchiature istofotometriche].[Article in Italian]. Riv Istoch Norm Pat 1954;1:149-70.
Vialli M, Zanotti L, Bianchi U. [A double histophotometer with direct vision for vision for visible and ultraviolet light].[Article in Italian]. Mikroskopie 1960;15:72-81.
Ornstein L. The distributional error in microspectrophotometry. Lab Invest 1952;1:250-65.
Adams LR. A photographic cytophotometric method which avoids distributional error. Acta Cytol 1968;12:3-8.
Chieco P, Jonker A, Melchiorri C, Vanni G, Van Noorden CJ. A user’s guide for avoiding errors in absorbance image cytometry: a review with original experimental observations. Histochem J 1994;26:1-19. DOI:
Deeley EM. An integrating microdensitometer for biological cells. J Sci Instrum 1955;31:263-7. DOI:
Benedetti PA, Viola-Magni MP. A scanning integrating histophotometer. J Sci Instrum 1966;43:141-3. DOI:
Prenna G, Mazzini G, Cova S. Methodological and instrumentational aspects of cytofluorometry. Histochem J 1974;6:259-78. DOI:
Cova S, Prenna G, Mazzini G. Digital microscpectrofluorometry by multichannel scaling and single photon detection. Histochem J 1974;6:279-99. DOI:
Prenna G, Leiva S, Mazzini G. Quantitation of DNA by cytofluorometry of the conventional Feulgen reaction. Histochem J 1974;6:467-89. DOI:
Itikawa O, Ogura Y. Simplified manufacture and histochemical use of the Schiff reagent. Stain Technol 1954;29:9-11. DOI:
Van Duijn P. A histochemical specific thionine-SO2 reagent and its use in a bi-color method for deoxyribonucleic acid and periodic acid Schiff positive substances. J Histochem Cytochem 1956;4:55-63. DOI:
Kasten FH. Additional Schiff-type reagents for use in cytochemistry. Stain Technol 1958;33:39-45. DOI:
Kasten FH. The chemistry of Schiff’s reagent. Int Rev Cytol 1960;10:1-100.
Ploem JS. The use of vertical illuminator with interchangeable dichroic mirrors for fluorescence microscopy with incident light. Z Wiss Mikrosk 1967;68:129-42.
Prenna G, De Paoli AM. [La deidrotio-p-toluidina-SO2, un nuovo reagente tipo Schiff altamente fluorescente].[Article in Italian]. Riv Istoch Norm Pat 1964;10:185-6.
Prenna G, De Paoli AM. [Derivati tiazolici come reagenti tipo Schiff fluorescenti].[Article in Italian]. Rend Ist Lomb Sc Lett B 1964;98:267-73.
Prenna G, Bianchi UA. [Reazioni di Feulgen fluorescenti e loro possibilità citofluorometriche quantitative. 5) Citofotometria quantitativa in fluorescenza ed in assorbimento della reazione di Feulgen eseguita con acriflavina-SO2].[Article in Italian]. Riv Istoch Norm Pat 1964;10:667-76.
Prenna G, De Paoli AM.[ Impiego del Rivanol come reagente tipo Schiff fluorescente nella reazione di Feulgen].[Article in Italian]. Riv Istoch Norm Pat 1968;14:169-70.
Mazzini G, Bottiroli G, Prenna G. An electronic device for the automatic correction of fluorescence emission spectra. Histochem J 1975;7:291-7. DOI:
Kamentsky LA, Melamed MR, Derman H. Spectrophotometer:new instrument for ultrarapid cell analysis. Science 1969;150:630-1. DOI:
Van Dilla MA, Trujillo TT, Mullaney PF, Coulter JR. Cell microfluorometry: a method for rapid fluorescence measurement. Science1969;163:1213. DOI:
Laerum OD, Farsund T. Clinical applications of flow cytometry: A review. Cytometry 1981;2:1-13. DOI:
Robinson JP. Handbook of flow cytometry methods. New York: Wiley-Liss; 1993.
Shapiro HM. Practical Flow Cytometry. New York: Alan R Liss Inc.; 1998.
Göhde W. Automation of cytofluorometry by use of the impulsmicrophotometer. In: Thaer A, Sernetz M, eds. Fluorescence techniques in cell biology. New York: Springer-Verlag; 1972. DOI:
Trujillo TT, Van Dilla MA. Adaptation of the fluorescent Feulgen reaction to cells in suspension for flow microfluorometry. Acta Cytol 1972;16:26-30.
Prenna G, Mazzini G, Bottiroli G, Freitas I. Automated determination of DNA cellular content (Feulgen) improved by using BBT-SO2 in flow cytofluorometry. In: Göhde W, SchumannJ, Büchner T, eds. Flow-through cytophotometry, its application to cancer research and hematology. Ghent: European Press, Medikon; 1976. pp: 88-95.
Rigler R Jr. Microfluorometric characterization of intracellular nucleic acids and nucleo-proteins by Acridine Orange. Acta Physiol Scand 1966;67:1-122.
Darzynkiewicz Z. Acridine orange as a molecular probe in studies of nucleic acids. In: Melamed MR, Mullaney PF, Mendelsohn, eds. Flow cytometry and sorting. New York: J. Wiley & Sons; 1979.
Crissman HA, Steinkamp JA. Rapid simultaneous measurement of DNA, protein, and cell volume in single cells from large mammalian cell populations. J Cell Biol 1973;59:766-71. DOI:
Krishan A. Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 1975;66:188-93. DOI:
Fried J, Perez AG, Clarkson BD. Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data. J Cell Biol 1976;71:172-81. DOI:
Giordano M, Danova M, Riccardi A, Mazzini G. Simultaneous detection of cellular ras p21 oncogene product and DNA content by two-parameter flow cytometry. Anticancer Res 1989;9:799-803.
Danova M, Pellicciari C, Bottone MG, Gibelli N, Mangiarotti R, Zibera C, et al. Multiparametric assessment of the cell-cycle effects of tamoxifen on mcf-7 human breast cancer cells. Oncol Rep 1994;1:739-45. DOI:
Tavecchio M, Simone M, Bernasconi S, Tognon G, Mazzini G, Erba E. Multi-parametric flow cytometric cell cycle analysis using TO-PRO-3 iodide (TP3):detailed protocols. Acta Histochem 2008;110:232-44. DOI:
Ferrara F, Daverio R, Mazzini G, Bonini P, Banfi G. Automation of human sperm cell analysis by flow cytometry. Clin Chem 1997;43:801-7. DOI:
Danova M, Riccardi A, Mazzini G. Cell cycle-related proteins and flow cytometry. Haematologica 1990;75:252-64.
Mazzini G, Danova M. [Citometria a flusso: applicazioni cliniche dell’analisi del DNA in oncologia].[Book in Italian]. Genova: Scuola Superiore Oncologia e Scienze Biomediche; 1994.
Tirindelli Danesi D, Spanò M, Altavista P. Quality control study of the Italian Group of Cytometry on flow cytometry DNA content measurements: II. Factors affecting inter- and intralaboratory variability. Cytometry 1997;30:85-97. DOI:<85::AID-CYTO3>3.3.CO;2-3
Mazzini G, Giordano P, Riccardi A. Biological significance of flow cytometric application of phenantridinic dyes at low concentration. Bas Appl Histochem 1980;24:264.
Mazzini G, Giordano PA. Flow cytometry: a methodologic approach for fast quantitative cytochemical measurements and its use for the study of the chromatin structure. Bas Appl Histochem 1981;25:303.
Mazzini G, Ferrari C, Erba E. Dual excitation multi-fluorescence flow cytometry for detailed analyses of viability and apoptotic cell transition. Eur J Histochem 2003;47:289-98. DOI:
Pellicciari C, Danova M, Giordano M, Fuhrman Conti AM, Mazzini G, Wang E, et al. Expression of cell cycle related proteins proliferating cell nuclear antigen (PCNA) and statin during adaptation and de-adaptation of EUE cells to a hypertonic medium. Cell Prolif 1991;24:469-79. DOI:
Robinson JP, Roederer M. History of science. Flow cytometry strikes gold. Science 2015;350:739-40. DOI:

How to Cite

Mazzini, G. (2024). The Feulgen reaction: from pink-magenta to rainbow fluorescent at the Maffo Vialli’s School of Histochemistry. European Journal of Histochemistry, 68(1).