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Conn s biological stains a handbook of dyes stains and
fluorochromes for use in biology and medicine 10th
edition Edition Biological Stain Commission. Digital
Instant Download
Author(s): Biological Stain Commission.; Conn, Harold Joel; Horobin,
Richard W.; Kiernan, John Alan
ISBN(s): 9781003076841, 100307684X
Edition: 10th edition
File Details: PDF, 98.94 MB
Year: 2002
Language: english
 CONN'S
Biological Stains

AHandbook of Dyes, Stains and

Fluorochromes for Use in Biology
and Medicine
10th Edition
Purchasers of the printed version will be entitled to free online access for a
trial period, after which access will be limited to subscribers. The online
version will have regular updates. Purchasers of the print version should
register by sending an email to [email protected]. Notification will be
sent out when the online version is released.
 CONN'S
Biological Stains
AHandbook of Oyes, Stains and
Fluorochromes for Use in Biology
and Medicine
10th Edition

Edited by

Richard W. Horobin
Institute of Biomedical and Life Sciences, University of Glasgow, UK

and

John A. Kiernan
Department ofAnatomy and Cell Biology, University of Western Ontario, London,
Ontario, Canada

9...."
Taylor & Francis
Taylor & Francis Group

LONDON AND NEW YORK

© Biological Slain Commission, 2002

First published 1925

Second edition 1929
Third edition 1936
Fourth edition 1940
Fifth edition 1946
Sixth edition 1953
Seventh edition 1961
Eighth edition 1969
Ninth edition 1977
Tenth edition 2002

All rights reserved. No part of this book may be reproduced or transmitted, in

any form or by any means, without permission.

A CIP catalogue record for this book is available from the British Library.

ISBN 1 85996099 5

Published by Talyor & Francis

2 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN
270 Madison Ave, New York NY 10016

Transferred to Digital Printing 2008

Production Editor: Andrea Bosher

Typeset by Saxon Graphics Ltd, Derby, UK

Publisher's Note
The publisher has gone to great lengths to ensure the quality of this reprint
but points out that some imperfections in the original may be apparent.
 Contents
How to use this book vii
Contributors list ix
Preface x
Acknowledgements xiii
Dedications xv

1. Introduction to dyes and stains. F.H. Kasten 1

2. The history of staining. B. BracegirdZe 15
3. Nomenclature and classification of dyes and other
coloring agents. rA.
Kiernan 23
4. Applications of dyes, fluorochromes and pigments. M. Wainwright 41
5. Mechanisms of biological staining. R. W Horobin 53
6. Dye purity and dye standardization for biological staining. H. Lyon 67
7. Reactive staining reagents and fluorescent labels. rC Stockert 77
8. The use of dyes and fluorochromes as indicators. M. Wainwright 89
9. Nitroso and nitro dyes. R. W Horobin 101
10. Monoazo dyes. R. W Horobin 107
11. Dis-, tris- and polyazo dyes. R. W Horobin 125
12. Diazonium salts and their reaction products with coupling
agents. R.W Horobin 145
13. Tetrazolium salts and formazans. R. W Horobin 157
14. Amino di- and triarylmethane dyes. R. W Horobin 169
15. Hydroxy triarylmethanes. R. W Horobin 203
16. Xanthenes. R.W Horobin 219
17. Acridines and phenanthridines. R.W Horobin 253
18. Azines. R. W Horobin 267
19. Oxazines and related dyes. R. W Horobin 277
20. Thiazines. R. W Horobin 293
21. Romanowsky-Giemsa stains. D.H. Wittekind 303

v
Contents

22. Polyene dyes and fluorochromes. R.W Horobin 313

23. Polymethine dyes -1. Cyanines, oxonols, benzimidazoles,
indolenines and azamethines. R. W Horobin 323
24. Polymethine dyes ­ 2. Styryls, thiazoles, coumarins and
flavonoids. R. W Horobin 349
25. Carbonyl dyes including indigoids, anthraquinones and
naphthalimides. R. W Horobin 367
26. Phthalocyanines, porphyrins and related aza[18]annulenes.
R. W Horobin 379
27. Miscellaneous inorganic and organic substances used as
biological stains. J.A. Kiernan and R. W Horobin 389
28. Methods for testing biological stains. D.P. Penney and J.M. Powers,
with the assistance ofC Willis, M. Frank and C Churukian 417

Bibliography 427

Index 539

vi
 How to use this book

How to find entries for individual dyes and fluorochromes

Within any major chemical grouping, for which see Chapter 3, non-ionic
compounds are treated first, followed by anionic, then zwitterionic, cationic,
and finally reactive dyes. Within each subgroup the entries are set out in
increasing order of the dye's formula weight. This arrangement usually places
compounds with similar properties near to one another in the text. The Index
provides an even simpler route to substances whose names, or Colour Index
designations, are known.

How to find information about a named dye

Find the major entry for a dye or fluorochrome, as above. In each entry, when
available and appropriate, information is provided on the following topics, in
the sequence given:

Usual dye name

Colour Index designations
Synonyms
Empirical formula and formula weight

Absorption and emission maxima

Qualitative indicator properties and pK values
Metal complexation behavior
Solubilities, in various solvents

Sketch of chemistry of dye with a structural formula

Staining applications in biomedicine - routine, and less common. Microscopic

stains are emphasized, but stains for flow cytometry are also noted.
Other biomedical applications - e.g. macro stain, or use as a marker, tracer or
assay reagent

Industrial uses

vii
How to use this book

Purity of commercial batches - homogeneity, dye content (including the

minimum required for Commission certification), availability of HPLC or
thin layer chromatographic methods, note of Commission assay methods.

Stability - of dry powder & staining solutions; light fastness

Special hazards

How to find the stain you need

Now suppose you are asking yourself a question such as, "Which dye could I
use to counterstain nuclei yellow, after doing this blue immunostain?/1 Does this
book help here? Not directly; this is not a staining manual. The book will,
however, inform you where to go for such advice: to the listing of practical
sources found under the heading Literature of biological stains, at the end of
Chapter 1.

viii
 Contributors list
Or Brian Bracegirdle, Cold Aston Lodge, Cold Aston, Cheltenham GL54 3BN, UK
Or Richard W Horobin, Division of Neuroscience & Biomedical Systems,
Institute of Biomedical & Life Sciences, The University of Glasgow, Glasgow
G128QQ, UK
Or Frederick H. Kasten, Department of Anatomy & Cell Biology, James H .
Quillen College of Medicine, East Tennessee State University, P.O. Box
70582-0582, Johnson City, TN 70582, USA
Or John A. Kiernan, Department of Anatomy & Cell Biology, The University of
Western Ontario, London, Canada N6A 5C1
Or Hans O. Lyon, Department of Pathology, Hvidovre Hospital, University of
Copenhagen, Kettegard Alle 30, DK-2650 Hvidovre, Denmark
Or Oavid P. Penney, Biological Stain Commission, Department of Pathology
and Laboratory Medicine, University of Rochester Medical Center, Box 626,
Rochester, NY 14642-0001,USA
Or James M. Powers, Department of Pathology and Laboratory Medicine,
University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY
14642-0001, USA
Or Juan C. Stockert, Facultad de Ciencia, Departamento de Biologia, Universidad
Autonoma de Madrid, Cantoblanco, E-28049 Madrid, Spain
Or Mark Wainwright, Senior Research Fellow, Department of Colour Chemistry,
The University, Leeds LS2 9JT, UK
Professor Or Oietrich Wittekind, Anatomische Institut der Universitat,
Albertstrasse 17, 0-78000 Freiburg, Germany

ix
 Preface
There have been nine previous editions of Biological Stains, published from 1925
to 1977. In each of these books, chapters concerning general aspects of dyes and
other stains were followed by a compendium of more detailed accounts of indi­
vidual compounds, grouped in chapters on the basis of chemical structure. The
tenth edition has a similar plan, although it is a multiauthor work, for which all
the chapters have been newly written. The average reader is assumed to be a
biologist or from a biomedical field; but not a specialist in analytical, organic,
physical or dyestuff chemistry, and not an expert in animal or plant histology,
pathology or toxicology. The book is a source of information about dyes, fluoro­
chromes and other colorants; and an account of their uses. It does not contain
technical instructions, for which the reader must consult the manuals and
articles cited in the text.
Overview. The first two chapters provide respectively a sketch of staining in
biological laboratories and the history of staining for microscopy. Chapter 3
explains the categorization of dyes and fluorochromes, which differs from that
in previous editions, along with some principles that govern the spelling of
informal names of dyes. Applications, including those outside the laboratory,
are reviewed in Chapter 4; and this is followed by a chapter reviewing the mech­
anisms of attraction and attachment of dyes to biological substrates. Examples
and mechanisms of staining and labeling by covalent attachment are discussed
in Chapter 7, and dyes used as indicators are reviewed in Chapter 8.
Conn's Biological Stains is published for the Biological Stain Commission, an
independent non-profit organization in the USA that tests and certifies many
stains, especially those used in histopathology. Standardization is also
attempted elsewhere and by other organizations; this is described in Chapter 6,
together with an account of the impurities that occur in dyes - the raison d'etre
of the Biological Stain Commission. Most of the remaining 20 chapters contain
accounts of particular dyes and fluorochromes, and the final chapter relates to
the methods used by the assay laboratory of the Biological Stain Commission.
Included and excluded items. Dyes and fluorochromes have been chosen for
inclusion on the basis of being mentioned in recent published literature, both
staining manuals and research papers. Only substances of known chemical
composition are discussed. Consequently some proprietary colored and fluo­
rescent reagents used by research workers are not included. One of us discussed
this issue with a major supplier, with his persuasive efforts proving inadequate
when faced with commercial pressures. The ninth edition of Conn's Biological
Stains (Lillie, 1977) included several dyes, some of questionable identity, that
had not been available for many years, and also some newer ones that never

x
 Preface

came to be used as stains. Most of these items have been omitted from the tenth
edition to make room for newer compounds, especially fluorescent labels and
probes, currently widely used in laboratories.
The electronic version. To keep this book a reasonable size, about half the
recent material that was unearthed from the literature has been omitted. We
have also omitted entries for compounds that have been little used in recent
years, and we do not include many older but nevertheless useful references to
traditional uses of dyes. A complete and fully searchable version of Conn's
Biological Stains will be available on a web site to purchasers of the printed
book. This electronic version will cover more compounds, give more examples
of older and current uses and will be updated at regular intervals. Purchasers of
the printed version will be entitled to free online access for a trial period, after
which access will be limited to subscribers. The online version will have
regular updates. Purchasers of the print version should register by sending an
email to [email protected]. Notification will be sent out when the online
version is released.
Disclaimers. The inclusion of a substance in this book does not constitute a
commendation of its efficacy for any purpose. Note that dyes and fluo­
rochromes often vary in composition (see Chapter 6), and consequently there
are inconsistencies in phYSicochemical data such as solubility and absorption
maxima reported in the literature. The values given here should be taken as
indicative, not authoritative.
For the case of solubility an additional, and specific, caveat should be given.
Dye molecules stick to themselves, as well as to cells and tissues; resulting in
ready formation of colloidal suspensions and related forms . This makes meas­
urement of solubilities a technically difficult, indeed a conceptually ambiguous,
task. Solubility data deserve especial skepticism even in the, unusual, circum­
stance of their relating to pure dye samples.
Toxic, allergic and other hazards are mentioned for some, but not all items.
Although the great majority of dyes do not pose serious hazards, laboratory
workers must always follow good laboratory practices and obey the rules set
out by local safety officers and committees.
Most of the dyes discussed in this book are or formerly were items of
commerce, and many of their names are or were legally protected terms.
Biological and biomedical end-users and writers routinely ignore this. They will
refer, for instance, to Texas red without noting that this is a legally protected
term owned by Molecular Probes Inc., or to dye names containing the words
coomassie or sirius without regard to the Ciba-Geigy or Bayer corporations
respectively. Failure to mention the owner of a trademark does not imply any
disrespect for the originators of such names, without whom there would be no
biological stains.

xi
 Acknowledgements
As editors, we recognize the contribution of Or Fred Kasten, past President of the
Biological Stain Commission, who started the process of revising the ninth edition
of Conn's Biological Stains. We also appreciate the support of the Trustees of the
Commission during this book's six-year gestation.
Assistance of various kinds, including spectral measurements, have been
provided by many people, including: Russ Allison (University of Cardiff), Mike
Barer (University of Leicester), Dan Belliveau (University of Western Ontario),
Dick Dapson (Anatech Inc.), Dave Goldman (Research Software Design,
Portland, Oregon; author of the PAPYRUS bibliographic database program),
Andi Grantham (California), Floyd Green (formerly of Aldrich Inc.), John
Griffith (University of Leeds), Bryan Hewlett (McMaster University), Lamar
Jones (Histological Consultants Inc.), Andrew Moran (University of Sheffield),
Debbie Rai (University of Stirling), and Juan Stockert (Free University of
Madrid), members of the staff of Aldridge Chemicals Inc. (Milwaukee, WI),
Amersham Pharmacia Biotech (Amersham, UK), and Molecular Probes Inc.
(Eugene, OR), and Mrs Denise Currie, Mrs EA Gordon and other librarians at
the Universities of Glasgow and Strathclyde. Mark Wainwright (University of
Leeds) and Tom Wickersham (formerly of Aldrich Inc.) provided valuable on­
going guidance to the complexities and peculiarities of dyestuff chemistry. We
thank all of them, and apologize to those others excluded only by the failures of
our notebooks.

May 2002 RW.H.

J.A.K.

xiii
 Dedications
From RWH:
To Dorothy, long after time;
To Rose, not before time;
and to The Trustees, hopefully just in time.

From JK:
To my family, especially Tessa, Julia, Edward,
Susan, Jeffrey and Philip, with love and appreciation.

xv
 The Biological Stain
Commission
Biological Stain Commission is a non-profit corporation, governed by a board of
Trustees, that has been testing and certifying dyes for use in biology since 1922.
Among the objectives of the Commission are the education of users about
sources of reliable dyes and fluorochromes, and the publication of information
about these substances and about traditional, improved and new staining
methods and histochemical techniques. These objectives are met by publishing
books and a journal. The first edition of Biological Stains by Harold J. Conn, a
founder of the Commission, was published in 1925. The Commission's
bimonthly journal was entitled Stain Technology from 1925-1990 and has been
Biotechnic & Histochemistry since 1991. The Commission's laboratory is
located in Rochester, NY. The web site at www.biostains.org may be consulted
for more information.

Trustees of the Biological Stain Commission, 2002

Graeme P. Berlyn, New Haven, CT
Alton Floyd, Edwardsburg, MI
William P. Grizzle, Birmingham, AL
Richard W. Horobin, Glasgow, Scotland
Frederick H. Kasten (emeritus), Johnson City, TN
John A. Kiernan, London, Canada
James R. Longley (emeritus), Louisville, KY
Hans Lyon, Hvidovre, Denmark
Robert W. Mowry, Birmingham, AL
G. Stephen Nettleton, Louisville, KY
David P. Penney, Rochester, NY
Robert Redman, Washington, DC
Clive R. Taylor, Los Angeles, CA
Dietrich Wittekind (emeritus), Freiburg, Germany
 IT]
Introduction to dyes and stains
F.H. Kasten

The first 8 chapters of Conn's Biological Stains review several aspects of the dyes
and other coloring agents used in botanical, biomedical, microbiological and
zoological laboratories. This first chapter introduces the reader to the termi­
nology, chemical natures, and some other properties of colored and otherwise
visible or potentially visible substances. Difficulties in obtaining correctly
labeled and sufficiently pure compounds are pointed out, and attempts to
address these problems are summarized. Chapter 1 concludes with lists of some
major books and journals that cover the large field of dyes, dyeing, biological
staining and histochemistry.

THE NATURE OF BIOLOGICAL STAINS AND THEIR USES

The purpose of staining is to add color to plant or animal tissues. This is done
sometimes to increase the visibility of objects (or regions within an object) that
can be seen with the unaided eye. The application of an iodine solution to a
piece of food is a traditional test for starch, which forms a blue complex with
iodine. In gynecological practice, iodine applied to a mucous membrane iden­
tifies and delimits areas of epithelium that contain glycogen. The red-brown
iodine-glycogen complex contrasts with the deep yellow color imparted by
iodine to almost everything else. There are many other macroscopic tests of this
type, but most of the rich variety of staining methods is due to the need to reveal
finer structural details of tissues and the more precise positions of chemical
substituents when viewed with a microscope.

Colored compounds and dyes

The great majority of substances used in biological staining are dyes. Almost all
dyes are organic compounds of the aromatic series and are therefore derivatives
of benzene, C"H". The six carbon atoms of benzene are joined to form a ring. The
C-C bonds in the ring can be illustrated in different ways, as shown in Figure 1.1.
Kekuh~'s representation, with alternating single and double bonds, is used in
the structural formulae of aromatic ring systems in this book because it shows

1
Conn's Biological Stains

Archaic Kekule's Formula emphasizing

representations of formula for equivalence of all
the benzene ring benzene carbon-carbon bonds

Naphthalene Anthracene Pyrazole Tetrazole

Thiazole Indole Quinoline Acridine

Xanthene Oxazine

Phenazine Phenothiazine

Figure 1.1 Representations of benzene (above) and some other aromatic ring systems
found in dye molecules.

the arrangements of atoms and bonds responsible for color more clearly than
the other conventions. Alternating double and single bonds are said to be
conjugated. The electrons that form the bonds are shared by the whole conju­
gated system (delocalized), and a structural formula showing single and
double bonds in certain positions represents only one extreme form that the
bonds may transiently assume. Aromatic compounds commonly contain two
or more fused rings (Figure 1.1), and one or more of the carbon atoms may be
replaced by another element (a heteroatom) such as nitrogen or sulfur. Some
examples that form parts of dye molecules are shown in the lower part of
Figure 1.1.

Chromophores and auxochromes

Benzene, naphthalene and similar aromatic hydrocarbons absorb radiation from
the ultraviolet but not from the visible part of the spectrum and are therefore not
calored. In order to absorb visible light the aromatic rings must form part of a

2
 Introduction to Dyes and Stains

larger molecule, known as a chromogen, in which one possible arrangement of

the conjugated bonds is not entirely aromatic. This results in an uneven distri-
bution of electric charge in the molecule. Examples of chromogens are given in
Figure 1.2. The part of a chromogen responsible for the property of color is
called a chromophore.
The notion of chromophores and chromogens was developed in 1876 by O.N.
Witt, 21 years before the discovery of the electron. It is now known that ultra-
violet and visible light are absorbed when photons interact with electrons, and
modern theories of color formation are consequently far more complicated than
indicated above (Harms, 1965; Horobin, 1982). A simple descriptive account
of the interaction of light with conjugated systems of electrons in organic
molecules is provided later in this chapter.
The colored compounds shown in Figure 1.2 are not dyes; they would not
stain a fabric or tissue. In order to be a dye a colored compound must be able to
impart its color to other substances. This property is achieved by adding to the
molecule one or more substituent groups known as auxochromes, which are
capable of joining the chromogen to the substance being dyed. Auxochromes
also modify (and usually increase the intensity of) the color of the chromogen.
The simplest auxochromes are side-chains that can form positive or negative
ions. Examples are amine groups (which are protonated in acidic media),
quaternary nitrogen atoms (which are positively charged at any pH), sulfonic
acid groups (negatively charged when dissolved in water at any pH), and
carboxyl groups (which ionize to yield negative ions in neutral or alkaline
conditions). Most solid dyestuffs are salts in which the auxochrome ions are
balanced by sodium or chloride. The charged dye ion is attracted to oppositely
charged ions that form part of the material being dyed. For example, negatively
charged dye ions, applied from an acidic solution, bind to protonated amino
groups of proteins, including those of cytoplasm and collagen in sections of
animal tissues, or the keratin and sericin of which wool and silk are respectively
composed. Attraction of opposite charges is one of several mechanisms that can
serve to bind a dye to a substrate. The mechanisms of staining are discussed in
detail in Chapter 5.

Light absorption by organic molecules

Absorption of ultraviolet or visible light is a function of the energy required to
promote electronic transitions within the molecule and there is now a sufficient

o-benzoquinone 1-nitronaphthalene azobenzene

quinonoid chromophore nitro chromophore azo chromophore

Figure 1.2 Some colored compounds (chromogens) that are not dyes.

3
Conn's Biological Stains

knowledge base to enable the prediction of light absorption for a given molecule,
normally using molecular orbital calculations. The Parr-Parriser-Pople (PPP)
method is perhaps the best known of these, but an excellent qualitative treatment
is given in the standard work on this subject by Griffiths (1984).
The presence of functionalized aromatic features (heteroatoms,
auxochromes), allows the absorption of visible light. The absence of part of the
perceived reflectance or transmittance spectrum gives the impression of a
certain color or shade. For example, removal of the red component of white
light gives the sensation of blue col or, with the shade of blue depending on the
exact type of red light (i.e. the wavelength range) absorbed. It should be
remembered here that there is a reciprocal relationship between wavelength
and energy.
Electronic absorption in organic molecules is allowed due to the presence of
vacant electronic orbitals (levels). Normally, the absorption of light energy
allows the promotion of an electron from the highest occupied molecular orbital
(HOMO) to the lowest unoccupied molecular orbital (LUMO, vacant). The
energy required for this transition, that is, the gap between the HOMO and
LUMO, depends on the structure of the molecule. There are other transitions
that occur, and the more complex the molecule, the greater is the number of
absorption bands. The present discussion must be limited to the longest wave­
length of absorption ("-"In ) and for deeper treatments the reader is referred to the
texts of Griffiths (1984) and Zollinger (1991, 1999).
As a simple example, anthracene (Figure 1.2) is an aromatic molecule of the
benzenoid class, containing three linear-fused benzene rings. The lowest
energy absorption (i.e. the longest wavelength) of anthracene occurs in the
ultraviolet range (300 nm). This absorption can be lowered in energy by the
inclusion in the ring system of a nitrogen atom in the central benzene ring,
giving the acridine molecule (Figure 1.1), which has a lowest energy
absorption of 340 nm. The introduction of the ring nitrogen changes the
electronic structure of the aromatic system, narrowing the gap between
the energy levels.
The attachment of other chemical groups or atoms to the exterior of the ring
system can also affect the electronic transition, the magnitude depending on the
nature of the group and also its position in the ring. Where there is electronic
communication (conjugation) between the group and a ring heteroatom or
another, remote, group the effect will be significant. Important groups in this
respect are usually strongly electron releasing (such as amino) or withdrawing
(such as nitro). Such groups are often termed auxochromes.
Applying this second criterion to the acridine system, electron-releasing
dimethylamino groups (-N(CH3)' ) fixed in positions 3- and 6- of the ring are
both strongly conjugated with the ring nitrogen and lower the lowest energy
electronic transition still further, to 488 nm. The resulting dye is acridine orange
(see Chapter 17).
The use of heteroatom substitution and / or auxochromes is essential in
tailoring dyes to fit staining requirements. Both these factors also affect fluores­
cence properties. In addition, an auxochromic moiety can be produced with

4
 Introduction to Dyes and Stains

associated desirable physicochemical properties such as increased lipophilicity,

or tethered reactive groups for chemical attachment to macromolecules such as
proteins or nucleic acids.

Dyes, stains and colorants

The question is often asked, 'What are dyes and how do they differ from fluoro­
chromes, dyestuffs, colorants, biological stains, and pigments?' We have already
given a brief chemical explanation of what a dye is and stated that it is an
organic aromatic molecule containing the requisite groups that provide visible
color and permit molecular binding to a material. The analogous word fluoro­
chrome refers to a dye that is capable of fluorescing, though some fluoro­
chromes absorb only in the ultraviolet and are therefore not colored. 'Dye' is
synonymous with' dyestuff'. The latter word is probably derived from the
German word Farbstoff ('color substance'), which is translated as dye. Farbstoff
dates back to the products of the great German dye industries of the late 19th
century and has been used widely ever since. It is a matter of custom as to
whether the words dye or dyestuff are used. Dyestuff is popular in industrial
circles and dye is favored by histologists and cytologists. Examples of dyes are
the natural dye hematoxylin and the synthetiC dye crystal violet.
The word colorant is applicable to any substance that is capable of coloring or
staining a material. Baker (1958) argued that this word should be the term of
choice for biologists when referring to any substance that stains tissues. In spite
of the cogent argument made by this influential writer, the word colorant is still
not in vogue among biologists and histotechnologists.
The word stain, employed as a noun, includes not only dyes but all the other
colorants used in biological work, and some of these compounds are not
colored. For example, Schiff's reagent is used in the Feulgen method for DNA
(Kasten, 1964) and in the periodic acid-Schiff (PAS) test for polysaccharides and
glycoproteins (McManus, 1946). Fluorescent brightening agents are chemically
similar to dyes but their absorption is largely in the near ultraviolet. Their
various uses in biology include vital fluorescent staining of calcification in
tissues (Rahn and Perren, 1970) and detection of fungi in material that is also
conventionally stained to show bacteria (Ruchel and Schaffrinski, 1999). Dyes or
fluorochromes are produced as final products in the techniques of enzyme histo­
chemistry, immunohistochemistry, and in situ hybridization

Inorganic reagents
Other colorless colorants include osmium tetroxide and silver nitrate, which are
readily reduced to insoluble osmium dioxide (black) and metallic silver (brown or
black), in a finely divided or colloidal state. Gold may also be deposited in tissues,
nearly always by reduction of the tetrachloroaurate ion of HAuCl 4 or NaAuCl4
(salts colloquially called'gold chloride'). The color of colloidal gold may be violet,
red or black, depending on the average particle size (Frens, 1973). These and other
inorganic reagents are used in many histochemical and traditional staining

5
Conn's Biological Stains

methods. The older methods are often called metal impregnations, from the belief
that the soluble compound selectively permeated certain parts of the block or
section of tissue and was then reduced in situ to the visible metal or lower oxide
(Gatenby and Beams, 1950; Gray, 1954).
The adjective argentaffin means possessing the capacity to reduce simple
silver ions (Ag+) or complex ions such as Ag(NH,)+, in the dark and without the
aid of a chemical reducing agent. This is a property of some substances,
including serotonin (5-hydroxytryptamine) and ascorbic acid that occur at high
concentration in certain cells (Lillie and Fullmer, 1976; Pearse, 1985). An argyro­
philic (or argyrophil) object in a tissue also reacts with deposition of silver
atoms, but these are too small and too widely separated to interrupt the passage
of light. They resemble, in this respect, the specks of silver that form the latent
image in an exposed but undeveloped photographic film. A chemical reducing
agent (or exposure to bright light in a few methods) is required to produce a
black deposit of colloidal metallic silver. The chemical mixtures used to make
argyrophilic materials visible are similar to photographic developers. They
extract loosely bound (unreduced) silver ions from nearby parts of the tissue
and reduce them to the metal at the sites of previously formed invisible clumps
of a few silver atoms. Each tiny cluster of silver atoms serves as a catalyst, and
the silver particles grow until the original catalytic sites are big enough to be
seen. This is an example of amplification: the original signal is too small to be
detected, but it triggers other reactions that lead to a visible product.
The silver methods are the basis for the demonstration in light microscopy
of reticular fibers, intercellular boundaries, enterochromaffin (argentaffin)
cells, and the Golgi apparatus. Traditional neurological staining methods
depend heavily on the use of silver salts selectively to impregnate different
types of neuroglial cells and neurons and their cytoplasmic extensions.
Examples of such neurological methods are those named after Golgi, Ramon y
Cajal and Bielschowsky.

Electron stains
Salts of heavy metals, notably lead and uranium, are also used as electron stains,
to impart additional electron density to thin sections prior to examination by
electron microscopy. Electron stains are outside the scope of this book, but
considerable information about them is available elsewhere (Glauert and Lewis,
1998; Hayat, 1975; Horobin, 1982; Lewis and Knight, 1977; Ogawa and Barka,
1992; Pearse, 1985; Polak and Van Noorden, 1997; Stoward and Pearse, 1991).

Pigments
Another group of colorants is that of the pigments, defined for industrial
purposes as colored or white inorganic or organic substances consisting of small
particles, which are insoluble in the medium they are dispersed in, and which
remain insoluble throughout the coloration process. Pigments are used in
paints, inks and cosmetics, for bulk coloring of plastics, cellulose fibers for the

6
 Introduction to Dyes and Stains

paper industry, and many other materials. Inorganic pigments include titanium
dioxide (white), carbon (black), cadmium sulfide (yellow) and iron oxides
(yellow, red) (Schiek, 1982). Organic pigments are also widely used in industry
and include compounds representative of all the major chemical groups of dyes
(Singer, 1982).
Organic and inorganic pigments are formed during various biological
staining procedures. Examples are Prussian blue (in the Perls method for tissue
iron) and various azo, formazan and indigo compounds in histochemical tech­
niques for enzymes.

Dyeing and staining

Textile dye specialists, formerly called dyers, refer to the process of producing
permanent colors on natural and synthetic fibers as dyeing. While some biolo­
gists also use this word for their applications, most prefer to describe the
process of using a dye in solution as staining. Examples are hematoxylin and
eosin staining (which colors the nuclei of cells blue-purple and other tissue
components pink) and methyl green-pyronine staining, which imparts
different colors to DNA and RNA. These are techniques for use with sections of
fixed tissue.
Biological staining usually requires a prior fixation, which preserves the
architecture of the tissue and also enhances its affinity for some colored
compounds. This is not always the case, however, and nontoxic vital staining of
living cells has a long history, beginning with the use of carmine by Trembley in
the 18th century.
Vital staining is defined as the coloration of living cells or other components
of tissues (Emmel and Cowdry, 1964). When this is done in a whole living
animal or plant the term intravital staining has been used (Wilson, 1910).
Supravital staining is the application of dyes to living cells or small pieces of
tissue freshly removed from the body, which are believed to be alive at least
during the early stages of the procedure (Cappell, 1929). This type of staining
has been much used to study blood and other cell suspensions (Cunningham
and Tompkins, 1938). Intravital fluorochroming of whole animals was intro­
duced by Ellinger and Hirt (1929), who examined the microcirculation. In recent
years fluorescent compounds have come greatly to outnumber conventional
dyes in vital staining applications, and they are typically described merely as
fluorescent probes.
Examples of vital stains dating from the 19th century and still in use are
neutral red, which is taken up into endocytotic vesicles or vacuoles, Janus green
B, which enters mitochondria, and methylene blue, which enters the thin cyto­
plasmic processes of neurons (Baker, 1958). These dyes can be used in either
intravital or supravital techniques. A special type of intravital staining is the
tracing of neural connections. Any of a wide variety of fluorescent dyes (Aschoff
and Hollander, 1982; Kuypers et al., 1977) or fluorescently or enzymatically
labeled macromolecules (Kristensson et al., 1971; Mesulam and Rosene, 1979;
Trojanowski, 1983) is injected at a localized site. The tracer is taken up by

7
Conn 's Biological Stains

synaptic terminals and transported retrogradely along axons to the cell bodies
of neurons, in which it accumulates and is detected. Some tracers are trans­
ported anterogradely, from cell bodies at the site of the injection to the synaptic
terminals of the axons.
Certain anionic dyes such as trypan blue pass through damaged but not
through intact cell membranes and are then trapped in the cytoplasm. These
dyes therefore stain dead but not living cells. The trypan blue dye-exclusion
technique is a popular method used by cell culturists to quantify the proportion
of live and dead cells in freshly isolated cells. Several fluorochromes, including
propidium iodide, similarly enter dead cells but then bind strongly to the DNA
in their nuclei. Living cells can be selectively stained by immersion in a solution
containing an invisible compound that enters the cell and is changed to a fluo­
rescent substance as a result of an enzyme-catalyzed reaction. Fluorochrome
precursors of this kind include various esters and amides (acted upon by
esterases or peptidases) (Haugland, 1995; Johnson, 1998) and certain tetra­
zolium salts that change into colored or fluorescent formazans when they accept
electrons from coenzymes involved in cellular respiration (Sasaki and Passaniti,
1998; Ullrich et al., 1996). Thus vital staining provides a variety of ways to
determine the proportions of live and dead cells in suspensions and cultures.

NOMENCLATURE AND IDENTIFICATION OF DYES

The common names of dyes are meaningless and are not sufficient to identify
particular compounds. An individual dye may have several synonyms,
including the trade names, often long obsolete, of different manufacturers or
suppliers. Conversely, the same common name may be applied to completely
different dyes. Furthermore, a bottle may contain a substance completely
different from the dye named on its label.
Here is a simple example of difficulty in accurate dye identification. The
acridine dye known today as acriflavine (Cl 46000) was called trypaflavine
many years ago. This name does not appear in modern catalogs, so without
more information at hand a dye user would find it difficult to replace an old,
depleted bottle of trypaflavine under the old name. A listing of dye synonyms,
such as is provided in the index of this book, might solve this particular problem
but is not foolproof. Therefore, special systems of identification and numbering
are employed to verify the identity and chemical structure of each dye.

The Colour Index (Cl)

The Society of Dyers and Colourists (Bradford, UK) and the American
Association of Textile Chemists and Colorists (Lowell, Massachusetts) have
cooperated in identifying the chemical constitutions of most dyes. These are
listed under various classifications in six volumes of the third edition of the
Colour Index (1971-1975). Since then, three supplemental volumes have been
published, the last in 1992. The first digit of a page number in the Cl is the

8
 Introduction to Dyes and Stains

number of the volume. Copies of the Colour Index may be found as reference
books in university, technical, industrial, and government libraries. The work is
also available on CD-ROM as The Colour Index International (Society of Dyers and
Colourists, 1996), which is updated at frequent intervals. Since 2001 the 4th
edition of the Colour Index has been an online publication (information at
http:// www.colour-index.org).
Approximately 8000 individual dyes and pigments were initially identified
chemically and each was assigned a Cl generic name and a 5-digit Cl consti­
tution number to accompany its chemical structure. Both the Cl number and the
generic name provide an exact identification of a dye. The generic names are
usually less important to biological users because they are derived from modes
of application in textile dyeing (Acid dyes, Basic dyes, Direct dyes, Food dyes,
Reactive dyes, Natural dyes, Solvent dyes, Pigments, etc.). The Cl generic names
are found in sequence under their classification groups in Volumes I, 2, 3, 5, and
6 of the Colour Index. For example, methyl blue (Cl 42780) has the assigned
generic name Acid blue 93 (Vol. I, p. 1331). In the absence of a common name for
a dye, the Cl generic name is used. Cl Direct red 75 (Vol. 2, p. 2128) specifies an
azo dye that is used as a biological stain.
Volumes 4 and 5 of the Colour Index are of special value to people who want to
gain detailed information about dyes for biological staining. Volume 4 lists all
dyes with assigned Cl numbers in sequence, according to their chemical consti­
tution. For example, all xanthenes are listed by number, starting at Cl 45000 with
acridine red 3B and Cl 45005 for Pyronine C, and ending with Cl 45555
(Fluorescent brightener 155, a compound formerly added to lubricating oils). A
copy of a page from Volume 4 is shown in Figure 1.3. For each dye there is
recorded, in addition to the Cl number, the structural formula (with archaic
representation of aromatic rings), methods of preparation, chemical and
physical properties, references, patent numbers, and, where appropriate,
common names. Volume 5 is useful if one has a commercial name for a dye and
is looking for the Cl number. All commercial names are listed alphabetically. For
the example of trypaflavine, this name is found on p. 5787 of Vol. 5. Its Cl
Number is given as 46000. If one now looks in Volume 4 at Cl 46000, details are
given about acriflavine, the name now commonly used for this dye.

The Chemical Abstract Service (CAS) Registry

Precise identification of a dye may also be accomplished by reference to its
Chemical Abstract Service Registry or CAS number. These are unique numbers
assigned to compounds and mixtures recorded in the CAS System. The CAS
number is not related to chemical structure or composition, but is assigned to
each substance as it enters the registry. Every substance that has been cited in
chemical, scientific, and technical literature and has been indexed in Chemical
Abstracts since 1975 has been assigned a CAS number. This number is specific to
all details of a compound. It is therefore possible to specify a substance by way
of its CAS number, without regard to synonyms, trade names, or common
names. For the dye acriflavine, the CAS number is 8048-52-0.

9
Conn's Biological Stains

XANTHENE COLOURING MATTERS

The chromophore of the aminoXlUlthene dyes is the resonance-hvbrid rA:-Q

· J" IN /R
'R - ~O·/a
c -N '8' where
c
- V-a-'J' 8
R = H. or aIlr.yl. or aryl; the hydroxyxanthenes can be stabilised by the Iou of a proton. forming an uncharged system
in which the chromophore is the quinoid structure 0~:()=o
The dyes are prepared from unthene derivatives with the uoual auxochromes in ,......-poeition to the methane carbon atom.
Th... deriv.tives are not obtained from unthene itself. but by reacting together suitably choaen limple intermediatea.
When R is an aryl radical. the dyes. although po-mg the pyrone ring. have analogies with the triarylmethane cIaIL
The unthene clau il lubdivided into amino. aminohydroxy. and hydroxy derivatives.
[n general. the nothenea are buic dyes which poeaeoa remarkably pure bright hues. and their IOlutiolll are .transly
fiuOrescenL They dye wool and silk directly from weak acid baths. and cotton on I tannin mordanL Some of the hydroxy
compounds are valuable mordant dyes.
Special LitenNno
H.wilL Dymujf, dm-I tr- PyriIiiu. QIIiN>IiM. AcriIIiM. IJIfIt XiIIIIINM. Longmans. Green .It Co. London, 1922
Fierz-D.vid, KiUutlidu OrrtIIfiWN Farlntoff., Juliua Springu. Berlin, 1926
E[demeld, H.~ C~, Vol. 2. p. 419. John Wiley.lt So.... New York, 19S1
Venlwaraman, TM Chnrimy of IM SyootIutic Dy'" p. 740. Academic Preoo, New York, 19S2
Lubs, TM Chnrimy of SyootIutic Dy.,1JIfIt Pipau. p. 291. ReiDhold Publishing Corporation. New York, 19S5

XANTHENE COLOURING MATTERS

(1)- AMINO-DERIVATIVES (FLUORENE COLOURING MA'I"I'ERS)

(..) PyroaiDeo (CI.4SIIIlO-4S020) (~ RaamiDes (C.I••S09O-4SIOS)
(6) SucciDeins (C.I.•SOSO) (e) RhodamiDea (C.I••SIS()....4S22S)
(~) Soccbareina (C.I••S070)
(IJ) - AMINO·HYDROXY.DERIVATIVES (RHODOLS)
(IJI) - HYDROXY·DERIVATIVES (FLUORONE COLOURING MA'I"I'ERS)
(..) Hydroxy·phthaleina (C.I••S3S()....4S460) (6) Anthrahydroxy·phthaldna (C.I••SS0()....4SSIO)

_.....
([V) - MISCELLANEOUS.DERIVATIVES

(I) - AMINO-DERIVATIVES (FLUORENE COLOURlNG MATI'ERS)

(..) PYRONINES
o;-wr- _ _
-uooo -0,-
~1191

t-bonIc Co.. BP 1231/92; USP 489625; FP 219Oll; GP 65212

(ILCIB:N0~OKmCHolICt (Fr. 3. 176)
- . . . J. "..,. 0 - . 54 (1196). 235

OxicliM c.x.uo. with pow-,urn pcnnanpnaIII SoIubIoin.-

9aIubIo in _(NIl with _ _ ,.u.... f t _ I
_ _ NIl
H.SO. (NII ...... ..-,.u.... I - I
caac. -yoIIow with . . - _ ; on c l i l _ ­

" - 001_ + N.OH - NIl pp&.

45005 -0,­ ~-_I'19

"'-G~I
(ILC),~ON(CIL),ICJ .,... Co.. BP 1673/19; F,. 191715; GP 54190 (F•• 2, 61)
t-bonIceo..B,.U217/19.1_/9I; USP445684; FP ZOO401;
GP 51955. 59003. 6lOI1. (F•• 3, 92, 94, 9J)
Cond_ _ _ ~ wi... _Y'la.dabydraoooba Gort.ar Co., G,. 60505 (Fr. 3. 96)
product with sul.Nric acid. and o&id.iM with ferric c:hIoride BlOS959.IO
M __ xi. 4 [4) (Inol. 751
_ • K.icIo. /Mr. rt (11941. 2I9t
~. IW. rt (1194). 3199; J. "..,. 0-.54 (11961. 217
_ . F....... :n.M~S_ N"". 1924
H,so........ - racIcIiob yoIIow; on dilution - NIl
A _ ooIu_ + HCI- bftthc_ SoIubIain.-
SaIubIo ia _ ( N l l w i t h , - _ )
(NIl w i t h , . u . . . . - I

Figure 1.3 A typical page from the Colour Index showing er numbers, textile uses,
common names, chemical structures and other information. (Reproduced with
permission, from Vol. 4 of 3rd edn, p. 4417.)

10
 Introduction to Dyes and Stains

Substitution and rnisidentification of dyes

The label on a bottle does not always correctly name the contents. For example,
an investigation of pyronine dyes from various commercial sources (Kasten,
1962) revealed widespread substitution of rhodamines for pyronine Y(G) (Cl
45005), a dye widely used in a staining technique for nucleic acids. This situation
is much improved today as a result of a purer pyronine Y(G) supplied by
industry and the modern dye standardization procedures used by the Biological
Stain Commission (BSC) for dye certification. Confusion and deception about
the names of dyes used in biology were more widespread years ago but have
occurred as recently as the 1990s (Penney and Powers, 1995), when some
companies supplied unsatisfactory and unrelated products in place of light
green SF yellowish (Cl 42095). This dye is an ingredient of Papanicolau's stain,
used worldwide for detection of malignant cells in smears.
An interesting example of beneficial dye substitution is seen today in the case
of aniline blue WS (Cl 42755). This anionic triphenylmethane dye is a
component of many trichrome recipes to stain collagen. Aniline blue WS is often
contaminated with other dyes, including methyl blue (Cl 42780), which gives
equally good staining results, but older samples varied in their dye content and
staining properties. It is prohibitively expensive to purify aniline blue WS, but
relatively easy to synthesize methyl blue of satisfactory purity, and the latter is
now supplied by at least one major dye firm, as a certified dye but under the
name of aniline blue (Green, 1990). The company is to be commended for being
frank about this desirable substitution. Histologists and technologists need to
realize that the two dyes are interchangeable in trichrome procedures. More
than 20 names have been applied in the past to aniline blue WS, methyl blue and
mixtures of these and similar dyes (Krause, 1926-1927; Lillie, 1977).

LITERATURE OF BIOLOGICAL STAINS

There are numerous sources of information about the chemistry of dyes and
pigments. Useful references are the third edition of the Colour Index (Society of
Dyers and Colourists, 1971-1992; 1996) and works by:

Fay (1911)
Bucherer (1914)
Cain and Thorpe (1923)
Schultz (1931-1939)
Fierz-David (1949)
Venkataraman (1952-1978)
Lubs (1955)
Allen (1971)
Rys and Zollinger (1972)
Bird and Boston (1975)
Venkataraman (1977)

11
Conn's Biological Stains

Fabian and Hartman (1980)

Gordon and Gregory (1983)
McLaren (1986)
Waring and Hallas (1990)
Peters and Freeman (1991).
Zollinger (1991)
Zollinger (1999)

The previous editions of Biological Stains provide a historical view, reflecting

as they do the available information and interests for early periods in this field.
The first edition by HJ. Conn was published in 1925 and the ninth, by R.D.
Lillie, appeared in 1977. Other books with information about stains and
discussion of how they work include the following:

Mann (1902)
Becher (1921)
Krause (1926-1927)
Baker (1958, 1966)
Emmel and Cowdry (1964)
Harms (1965)
GUff (1971)
Gabe (1976)
Lillie and Fullmer (1976)
Horobin (1982,1988)
Clark and Kasten (1983)
Lyon (1991)
Hayat (1993)
Kiernan (1999).
Bancroft and Gamble (2002)

The Merck Index (Budavari, 1996) is a valuable compilation of information

about thousands of compounds, including many that are dyes or other
compounds used in staining. The catalogs and handbooks of some major
chemical companies contain valuable information about dyes such as the
Sigma-Aldrich Handbook of Stains, Dyes and Indicators (Green, 1990). A compre­
hensive source of data about fluorescent molecular probes is to be found in the
Handbook ofFluorescent Probes and Research Chemicals from Molecular Probes, Inc.
(Haugland, 1996). This company also has an updated CD-ROM that appeared
in 2000. A volume that centers particular attention on the theories of fluores­
cence, fluorescent dyes and probes, and on molecular interactions is Fluorescent
Probes in Cellular and Molecular Biology (Slavik, 1994). There are some compre­
hensive reviews of the history of fluorescent dyes/ probes and fluorescence
microscopy, as well as histological and cytological applications (Kasten, 1983,
1989, 1999). Other important books are available that focus on fluorescent dyes
and probes and their applications in biology. These volumes include
Fluorescence Microscopy of Living Cells in Culture (Part A edited by Wang and

12
 Introduction to Dyes and Stains

Taylor, 1989; Part B edited by Wang, 1989), Cell Structure and Function by
Microspectrofluorometry (edited by Kohen and Hirschberg, 1989), Flow Cytometry
(edited by Darzynkiewicz and Crissman, 1990) and Fluorescent and Luminescent
Probes for Biological Activity: A Practical Guide to Technology for Quantitative Real­
Tim e Analysis (edited by Mason, 1999).
For those engaged practically in the application of biological stains in
medicine, cell and molecular biology, histotechnology, histochemistry, and
immunostaining, many books from the older and recent literature are worth
knowing about. Some of the older volumes have historically valuable infor­
mation and useful stain recipes that do not appear in more recently published
books. The following list contains only a selection of the many books that
contain instructions for carrying out staining procedures:

Chamberlain (1901)
Mann (1902)
Ehrlich et al. (1903)
Guyer (1906)
Langeron (1925)
Krause (1926-1927)
Mallory (1938)
Johansen (1940)
Gatenby and Beams (1950)
Jones (1950)
Gray (1954)
Ganter and JollE$ (1969,1970)
Lison (1960)
McManus and Mowry (1960)
Thompson (1966)
Luna (1968)
Romeis (1968)
Gabe (1976)
Berlyn and Miksche (1976)
Lillie and Fullmer (1976)
Lewis and Knight (1977)
Humason (1979)
Drury and Wallington (1980)
Pearse (1980,1985)
Clark (1981)
Culling et al. (1985)
Boon and Drijver (1986)
Sheehan and Hrapchak (1987)
Polak and Van Noorden (1997)
Larsson (1988)
Stamp and Wright (1990)
Chayen and Bitensky (1991)
Armed Forces Institute of Pathology (1992)

13
Conn's Biological Stains

Stoward and Pearse (1991)

Van Noorden and Frederiks (1992)
Sanderson (1994)
Taylor (1994)
Presnell and Schreibman (1997)
Horobin and Bancroft (1998)
Kiernan (1999)
Ruzin (1999)
Bancroft and Gamble (2002)

The works of Gatenby and Beams (1950), Jones (1950) and especially Gray
(1954) provide comprehensive accounts of staining methods available at a time
when histochemistry was in its infancy. The three volumes of the 4th edition of
Histochemistry: Theoretical and Applied (Pearse, 1980, 1985; Stoward and Pearse,
1991) contain the most extensive available accounts of histochemical techniques
of all kinds.
It should also be mentioned that there are more than 60 volumes published by
Gustav Fischer on topics in histochemistry and cytochemistry under the general
title of Progress in Histochemistry and Cytochemistry.
Several journals regularly contain articles dealing with the chemistry and
industrial applications of dyes, and with the use of these compounds in
histotechnology and histochemistry. A listing of the more important journals
includes the following:

Acta Histochemica
Advances in Colour Science and Technology
American Journal ofClinical Pathology
Archives ofPatholoqy and Laboratory Medicine
Biotechnic and Histochemistry (formerly Stain Technology)
Coloration Technology (formerly Journal of the Society of Dyers and Colourists)
Cytometry
Dyer
Dyes and Pigments
European Journal ofHistochemistry (formerly Basic and Applied Histochemistry)
Histochemical Journal
Histochemistry and Cell Biology (formerly Histochemistry, and before that
Histochemie)
Industrial and Engineering Chemistry
Journal of Clinical Microbiology
Journal ofClinical Pathology
Journal ofHistochemistry and Cytochemistry
Journal ofHistotechnology
Journal of Microscopy (formerly Journal of the Royal Microscopical Society)
Journal ofNeuroscience Methods

14
 [IJ
The history of staining
B. Bracegirdle

BEFORE STAINS

When a child, or anyone else new to a microscope, first looks at a specimen, he

puts it on the stage as best he can, shines a light on it, and views it by reflected
light. This is exactly how Robert Hooke looked at most of the specimens he illus­
trated so magnificently in his book Micrographia of 1665. He went on to describe
how he made what we would now call sections in various planes, how he
achieved a kind of darkground illumination, and even how he cleared some of
his specimens. It is a great pity that much of what he said was not understood
and thus not acted upon. His readers may well have looked at the pictures and
not his text!
We now know that the microscopical image must resolve required detail at
the required magnification, but it must also provide sufficient contrast to allow
the structure of the specimen to be observed. This contrast can nowadays be
obtained physically (by optical means), and/or chemically (usually by
coloring). It remains a salutary lesson, however, to take a piece of fresh tissue
and observe it squashed very flat without staining, as would have been the
norm until at least the 1850s. A surprising amount of detail can be made out,
given a lot of patience and the odd tilt of the mirror.
Nonetheless, observers from the dawn of microscopy on must have wished for
more contrast in transparent specimens, increasingly so as magnifications slowly
climbed in the 18th- and rapidly in the 19th century. Leeuwenhoek followed Hooke
as a microscopist, and used the smear technique with fresh specimens, among
other techniques (most of which he kept to himself). However, scientific work after
his in the 18th century (and on into the 19th) was based almost entirely on injection.
Such injected preparations, often using several colors in different vessels, are quite
amazing when viewed, but do not tell very much about the nature of the specimen
apart from the course of the fine vessels. They took the average microscopical
observer by storm, so amazing do they look, and for sheer effect are still worth a
look if some can be found in old collections. A few 18th-century workers, such as
Hill, avoided such techniques, and used cochineal as a stain. Hill also invented the
microtome and used microincineration of plant sections to increase contrast. His
work represented a real scientific advance, but it also failed to take root.

15
Conn 's Biological Stains

Much more than this sample of results was obtained before 1830, of course,
but is outside the scope of this short chapter. More detailed accounts, including
one of the development of the microtome, have been provided elsewhere
(Bracegirdle, 1986; Clark and Kasten, 1983).

AFTER 1830

This date has been chosen because Canada balsam was introduced as a mountant
about then. Soon afterwards specimens began to be cleared on the slide before
mounting, and thus the basis of modern technique had been established,
although it would be 50 years before it became fully developed. The use of stains
at this time was just beginning to be accepted, and received its real impetus not
from scientific work as such, but from the sale of preparations for the growing
number of amateur microscopists; visibility of the specimen was paramount for
such a purpose. From about 1840 a vast number of slides was produced (the 3 xI
inch size was formally adopted late in 1839), and many still survive to illustrate
the development of staining. Older collections are well worth the attention of the
professional microscopist, for much has been reinvented several times.
Something must be said of techniques other than actual staining. The prepa­
ration of microscopic mounts is an involved procedure, and it is a relatively
modern concept that the prepared specimen might bear little resemblance to the
original condition, but that when a tissue is prepared in a wide variety of ways,
a full idea of its make-up can be obtained. It can be difficult to be sure just what
was done in earlier days, as methods were often only vaguely specified,
reagents might have contained significant impurities, and their names might be
quite different nowadays.
The method used to kill the animal providing the tissue can critically affect the
result on the slide, and heavy narcotization before sacrifice remains unhelpful.
The preservation of the tissue sample also affects the result. It was not until 1833
that chromic acid was first used as a hardening agent, although Malpighi had
hardened brains for study by boiling them as early as 1666. Mercuric chloride
was first used as a fixative in 1846, but not established until 1878. Acetic acid had
been used, as vinegar, to preserve food, and alcohol was known to have preser­
vative properties, but neither was used effectively in microscopy before the mid­
19th century. Osmium tetroxide ('osmic acid') was introduced as a fixative in
1864, but did not find widespread use until 1882, and formaldehyde was not so
used until 1893. References for all these are given by Bracegirdle (1986), but two
important points have emerged. One is that the date of first use might well be
very different from the date when the technique became widely used. The other
is that most of the dates of first use are surprisingly recent.

NATURAL STAINS

Saffron was used by Leeuwenhoek in 1714 to color muscle fibers (van

Leeuwenhoek, 1719). Such a use repeated nowadays shows that it would not

16
 The History of Staining

have been very effective. It seems that Reichel (1758) used logwood, in simple
unmordanted solution, to stain plant tissues. Hill (1770) introduced cochineal,
and both hematoxylin and carmine remain in use even today.
Carmine was the only dye used for many years, but was not applied as a
nuclear stain until Corti (1851) so used it. Unfortunately he published in French
in an obscure German journal and his work was ignored. Von Gerlach (1858)
used greatly diluted ammonia-carmine overnight, and rapidly established the
technique among his students. Early workers thought that carmine stained
everything, and even Beale (1860) was led to quite wrong conclusions as to the
course of nerve fibers as a result: a salutary lesson even today.
Hematoxylin is an unsatisfactory stain in the absence of a mordant. It was
mentioned for the first time after Reichel by Quekett (1848), whose book is an
important landmark in the history of staining. It was Bohmer (1865) who first
used logwood with an alum mordant, and this revolutionized the use of hema­
toxylin. Some famous names in histology published their own mixtures
containing this stain, but its ripening was not understood until the mechanism
was described by Mayer (1891).
Many other natural stains - virtually every colored plant substance - were
tried out over the years; a characteristic of such papers is their distinctly
alchemical approach, and none was scientifically very useful.

ANILINE COLORS

The introduction of mauve, the first aniline dye, by Perkin in 1856 was a major
innovation not only for microscopy but for the entire chemical industry. It was
first used in staining by Beneke (1862), whose sample might well have been
from a different source and had slightly different properties. Waldeyer (1863)
used mauve, and also Paris blue and basic fuchsine, and in the same year Frey
(1863) used aniline blue, and Roberts (1863) used picric acid. For a decade (apart
from the introduction of indigocarmine by Chronszczewsky (1864» the initial
spate of aniline dyes as stains was over. However, in 1874 Ranvier (1874) used
cyanin, Zuppinger (1874) introduced iodine violet, and Lieberkuhn (1874)
adopted synthetic alizarin.
The way was then clear. All histologists could scan the dyemakers' lists, and
publish a paper, perhaps of indifferent quality, on the use of a new color. From
this date any survey of the literature must be highly selective.
Methyl violet was introduced by Cornil (1875), in an important paper which
also described for the first time the metachromatic effects of aniline dyes.
Another important paper of that year was by Hermann (1875), which brought to
the attention of biologists the principle of differentiating a stain. This was not the
first description, for it had been first mentioned by Schweigger-Deidel and
Gogiel in 1866, and again by Bottcher in 1868, but neither of these earlier investi­
gators established the technique.
Ehrlich introduced at least 12 stains. These include safranine (Ehrlich, 1877),
which had been available since 1859 and remains a valuable stain. He also

17
Conn's Biological Stains

surveyed the then available dyes, making some mistakes as to their compo­
sition. He introduced methyl blue, nigrosin, and acid fuchsin in 1879, neutral
red in 1893, and janus green in 1898. His work with methylene blue (Ehrlich,
1881) was fundamental, especially for bacteriology.
Other significant stains introduced in this golden period included bismarck
brown (Weigert, 1878), malachite green (van Beneden and Julin, 1884), light
green and congo red (Griesbach, 1886), and the fat stains Sudan III (Daddi, 1896)
and Sudan IV (Michaelis, 1901).

MULTIPLE STAINING

The first double stain was that of Schwartz (1867), who used successive solu­
tions of picric acid and carmine. His paper has excellent color plates illustrating
the effects obtained. The first to counterstain hematoxylin with an aniline dye
was Poole (1875), who used aniline blue for the purpose. The following
year saw the introduction of the still widely-used hematoxylin and eosin
(Wissowzky, 1876). The first account of a triple stain was by Gibbes (1880), and
Griesbach (1889) demonstrated quadruple staining in 1888. Such techniques are
still in use for teaching, but it remains a truism that little is to be discovered
merely by the use of extra colors on one tissue.
Be that as it may, some combinations have stood the test of time. Van Gieson's
(1889) stain was originally worked out for nervous tissue but is now applied to
connective tissue. Flemming's (1891) triple stain is still used for nuclear
cytology, and Mallory's (1900) method is still valuable for showing collagenous
and cytoplasmic fibers in contrasting colors.
The blood stains are important, not only for their practical utility but also for
their scientific background. In their developed form they are combinations of
basic with acidic dyestuffs, and were originated by Ehrlich (1879), with some
understanding of their chemistry. Many others were to contribute to the
perfection of such stains, culminating in Giemsa (1902a,b), who made the stains
reliable. Bernthsen (1906) clarified the chemistry of the oxidation products of
methylene blue present in these stains.

THE BEGINNINGS OF CYTOLOGICAL STAINING

The mitochondrion was one of the first organelles to attract attention, and was
described under many different names before Benda (1901) described a
procedure that rendered results easy to obtain. After refinement (Meves and
Duesberg, 1908), the technique has remained useful into modern times. Golgi
bodies attracted more attention still, because they were lost in routine fixation.
Their very existence was fiercely disputed (Kirkman and Severinghaus, 1938)
until use of the electron microscope proved that they were present.
The first silver impregnation technique (Krause, 1844) was used on pieces of
skin. Improvements by von Recklinghausen (1860) brought this type of method
into wider use.

18
 The History of Staining

Gold salts were first used by Cohnheim (1866). Osmium tetroxide was used
by Golgi from 1873, and much improved in his rapid method (Golgi, 1886),
which took only a week to carry through. His preparations (which survive in
Pavia) are important in the history of histology. Ramon y Cajal developed the
work further, especially for spinal ganglia, and his double method (Ramon y
Cajal, 1891) was a considerable advance. Much mumbo-jumbo was written
about these techniques, but Hill (1896) clarified the various formulae.

COLORING LIVING TISSUES

Although Trembley colored Hydra in 1744, the real interest in such work
developed only from about 1880 (Certes, 1881; Ranvier, 1975). The modern
development of true intravital staining took place when Bouffard (1906) injected
acid dyes and Goldmann (1909) used a colloidal dye, pyrrhol blue. Subsequent
workers placed much reliance on intravital techniques, especially in the
development of histochemistry.
Although Girod-Chantrans used a variety of reagents and tests to classify plants
as early as 1802, it was Raspail (1825) who founded histochemistry. He deliberately
set out to apply histochemical tests in his researches, and consolidated the work in
a famous essay (Raspail, 1830). The further history of histochemistry has been
outlined by Petersen (1941), Sandritter (1964) and Pearse (1980)
Nothing has been said in this short summary about the development of infil­
tration and embedding methods, and the slow perfection of microtomy.
However, by the end of the 19th century, much that would still be recognizable
in a laboratory for histology had been developed. Possibly the most substantial
work ever in the field of practical histology was the third edition of a book
edited, in its first edition, by Ehrlich, and first published in 1903 (Krause, 1923).
This first edition is a good summary of the state of histological work at the turn
of the century, while the three volumes of the 1926-27 edition contain
2444 pages, and have copious references to each entry. This is some measure not
only of the thoroughness of the authors, but of the development of the subject in
a relatively few years.
The story may be brought further up to date by a series of editions of a
substantial book edited originally by Arthur Bolles Lee, first published in 1885
(Lee, 1885), and reaching its 11th edition in 1950. These summarize most work of
any note in the field, although coverage is less full in the last edition.
A few more general works on the nature of staining had appeared, prominent
among which is that by Mann (1902). This seminal work gave a detailed scientific
background of the mechanisms underlying the various techniques, as well as
detailed notes on the different processes themselves.

INTO THE TWENTIETH CENTURY

By the end of the 19th century, the optics of the microscope had been developed
to near perfection by the use of apochromatic objectives applied on a sound

19
Conn's Biological Stains

theoretical basis. Photographic recording of results had become less of an art

and more of a science; most of the required mounting procedures were in place;
some development of micromanipulation had taken place, and a few had
looked at living material as well as fixed.
Evans and Schulemann (1915) introduced specialized acid dyes (trypan red,
trypan blue, vital red, Evans blue) which remain of much importance, and vital
staining was given a further boost in the 1920s. Simpson (1921) investigated the
reactions of living leukocytes to more than 200 dyes, and Sabin (1923) analyzed
human blood cells using neutral red and janus green B. Stockinger (1964)
brought this work more up to date in a useful monograph on vital staining and
fluorochroming of animal cells.

FLUORESCENCE MICROSCOPY

The fluorescence microscope, first used for histology and histopathology in the
1930s, is of the greatest importance in the 1990s. The techniques have developed
beyond all recognition over the previous 25 years. Immunofluorescence, for local­
izing specific antigens relies on it, and both histo- and cytochemistry have been
revolutionized by it. The distribution of macromolecules in the cell can be demon­
strated using secondary fluorochromes, and a wide range of these is now available.
The technique was introduced by Heimstadt and Lehmann between 1911
and 1913 (Heimstadt, 1911). It was not to become widely used for well over
another 40 years, although both Zeiss and Reichert offered suitable instru­
ments by 1915. Almost all the important work before about 1940 was carried
out in Germany; suitable reviews of what had been achieved are provided by
Metzner (1931) and Hamperl (1943). All this work established the instrumen­
tation, and especially the light sources and filter systems. The secondary fluo­
rescence techniques were then applied in medicine and life sciences. The work
caused much excitement in physiology, and a great deal was achieved before
World War II intervened.

POST-1950 DEVELOPMENTS

Since about 1950 the world of light microscopy has been transformed at its
cutting edges. Naturally, the larger part of histological microscopy remains that
of routine coloring of sections. Less of this is now carried out in university
departments for training medical and life sciences students, as histology now
gets much less attention in premedical and other courses than it did until about
1985. (Interestingly, in the United Kingdom, it became an actual statutory
requirement to include a considerable amount of practical and theoretical
histology in the medical curriculum in 1886. It was to last for almost exactly a
century.) Much routine histology is still carried out for pathological diagnosis,
and this may well continue for many years yet. For those carrying out research,
however, matters are now rather different.

20
 The History of Staining

Any historian working on the very recent past faces two difficulties. One is
that there is a great abundance of material, making it impossible to appraise all
that is to hand. The other is that, until some years have elapsed, it is impossible
to decide what is going to be significant and what is not. These same difficulties
apply to the history of staining; the literature is vast and the important
techniques of tomorrow are impossible to forecast.

INCREASING PRECISION IN THE 19805 AND 19905

Nucleic acids were discovered as long ago as 1869, in Tiibingen, by Miescher

(1871), who extracted them from pus and realized that they came from the nuclei
of cells. From that observational feat came much of today's amazing precision in
identifying the nature and geography of a wide range of cellular components.
Miescher relied on gravity to recover his separated nuclei from other cell
components; it would not be until 1934 that the centrifuge would be applied to
cell fractionation. By the time of his death in 1895, Miescher recognized that he
had founded biochemistry, but he never accepted that nuclein (chromatin) was
the chemical foundation of heredity.
Zacharias (1881) had actually already demonstrated that chromatin, chromo­
somes, and nuclein were essentially one and the same, and others completed
similar work. Kossel discovered adenine, thymine, and thymic acid, and he was
awarded the 1910 Nobel prize in medicine for his work on the chemistry of cell
nuclei. In spite of all this work, 47 years were to pass before it was categorically
stated by Gulland (1938) that plant and animal nucleic acids were accepted as
being one and the same, and that DNA and RNA were separate kinds found in
both plant and animal cells.
Feulgen and Rossenbeck (1924) had pioneered the first cytochemical reaction
for DNA and it was applied to the giant salivary gland chromosomes of some
Diptera by King and Beams (1934). The Feulgen reaction obviously filled a great
need for a reliable DNA! chromosome stain; by 1938 more than 400 references to
the DNA nuclear reaction had been published, and it has remained a valuable
tool through and beyond the 1950s.
Large numbers of workers have applied more modern techniques, and more
especially a wide range of specific fluorochromes, to chromosome banding
methods. Others have developed autoradiography, flow cytometry, and a range
of other techniques to investigating with ever greater precision just what is
where in the cell, and just what it is doing there. The electron microscope has
been developed as an analytical tool, and various new kinds of light microscope
now allow investigation of living cells more easily and precisely than ever
before. The application of powerful electronics to imaging, image enhancement,
and computer analysis has opened up still more possibilities. It would be
premature here to analyze what might be achieved with all these even over the
next 10 years.

21
 IT]
Nomenclature and
classification of dyes and
other coloring agents
].A. Kiernan

Most of this chapter is concerned with the chemical groups of dyes used in
biology. At the end is a short section on more complex substances: probes and
labeled compounds.

NAMING OF DYES

The formal chemical names of most dyes are so long that they are never used in
conversation or writing. Instead, each dye has one or more informal or trivial
names. In addition, many dyes have standardized Colour Index names based on
their original industrial uses and colors.

Informal names
Most informal names include the color, and some also indicate a major property or
the general class of compounds to which the dye belongs. For example, acridine
orange is a dye that contains the acridine ring structure. Letters at the ends of
names may indicate similar dyes with slightly different colors such as eosin Y and
eosin B. Words in the informal names of dyes are often trade-names (e.g. coomassie,
eriochrome, sirius) that bear no relation to chemistry or color but have entered the
language of scientists through common usage. Often an informal name gives no
information about the chemical constitution or colorant properties of a dye. The
names methyl blue or Congo red might apply to almost any blue or red compound,
and names like vesuvin and coriphosphine do not reveal even the colors.

Colour Index names and numbers

The Colour Index (Cl) and its supplements, published by the Society of Dyers and
Colourists (1971-1996), is an encyclopedic reference for dyes and other coloring
agents. It provides for each dye a unique number, related to chemical structure, and

23
Exploring the Variety of Random
Documents with Different Content
upon him. He does not allow himself to be snared by
the lure of vivid, brilliant language, nor by the
intoxicating problems of inner truths whose surface
he grazes. According to him the sum of all his work
has been but to “shift a few grains of sand upon the
shore” of knowledge, and it is useless for him to
endeavour to sound the mysteries of life; he has not
even learned—he does not even think it possible to
human knowledge to learn—“the last word
concerning a gnat.”

Does this imply that he has relapsed into scepticism;

that finally, in despair, he renounces the ambition of
his whole life, vitam impendere vero? By no means.
He has striven to attain it even beyond his strength.
[380]

When he considers himself incapable of adding

further volumes to his work he busies himself with
preparing a definitive edition, and in a touching
farewell to his beloved studies he declares that they
are so full of charm and unexplored marvels that
could he live several lives he would devote them all
to them without ever succeeding in “exhausting their
interest.”

There we have Fabre. After labouring all his life

without troubling about fame, ploughing his straight
furrow like his peasant forebears, like them, when the
night has come, he simply binds his sheaves with a
humble and profound realisation of the narrow limits
of his work as compared with the immensity of the
world and the infinite mystery of things.

It is a fine spectacle, that of the entomologist on the

summits of science, as of fame, raising himself, by
his humility, above both, and fully prepared, to return
to Him toward whom aspire those souls that have
attained the limit of human climbing:

O Jesu corona celsior

Et veritas sublimior.

[Contents]

III

Neither science nor fame could prevent him from

suffering. To begin with, there is [381]suffering
attaching to these, for all labour has its burden, all
light its shadow.
This none knew better than he whose genius was a
protracted patience and his life a hard-fought battle.
And as though it was his destiny to suffer to the end,
he did suffer still when the tardy hour of his fame had
struck. Was it not an ordeal still to be assailed by
visits and speeches when “nothing was left but rest
and silence”? How can a man delight in the incense
of his admirers when he is broken with fatigue?

To express this contrast, to show that all was not

unmixed joy in these flattering visits to the patriarch
of Sérignan, I will borrow the delicate brush of an
artist friend of Fabre’s:

Night falls upon Sérignan, serene, limpid, violet and amethyst.

The sounds of day fade one by one. Still a few distant hoots
from the horns of motor-cars flying along the dusty roads, or
the sound of a dog baying the new moon, which shows its
slender sickle on the horizon; sometimes, too, as though to
eclipse the first stars, a rocket roars, a prelude to the
fireworks which are about to conclude the apotheosis.… J. H.
Fabre, the hero of the fête, the lover of the Sphex, the Mantis,
the Dung-beetle, is very tired. Think of it—ninety years of age,
and almost ninety years of labour!… and [382]a world-wide
reputation to sustain … and visits to receive. To-day it was the
visit of a Minister and all the flies on the ministerial wheel. And
he had to return thanks, feeling upon him the eyes of the
reporters and the photographer’s lens. What an ordeal! Fabre
can hold out no longer!…
Do you not feel that the harvest of fame at ninety
years of age and after almost ninety years of labour
is perhaps even more painful than the harvest of
science in the ardour of youth?

Meditating upon his history, with its full days and

hours, Fabié, in a delightful flight of imagination,
shows us the harassed entomologist escaping from
the past to find himself alone with his thoughts and
his beloved insects. “He slips silently to the gate of
his harmas. There he lies down on a bank thickly
carpeted with lavender and withered couch-grass”.…
A few moments pass. His children intervene: “he is
relaxing himself, stretching himself, soothed, happy
as a little child.—‘But, father, you aren’t thinking!
When the dew is falling!’ ‘Ah, my children, why did
you wake me? I was having such a beautiful dream!’
For in his sleep he had entered into conversation
with the crickets of his native country-side.”

Fatigue of the body, weariness of the mind, [383]and a

breaking heart! Suffering pressed closely upon him at
the close of his days.

“It is better to be loved than to be celebrated,” said

Aubanel, the delicate poet of Avignon. As long as
Fabre had beside him his beloved brother, his adored
wife, and his darling children, he was at least
conscious of a kindly atmosphere of memories, and
of tenderness that made up for what he lacked and
helped him to endure his afflictions with serene
resignation.

But now, little by little, there came a void about him.

Death has its surprises and life its demands.

With the death of his wife, in July 1912, half his own
soul died. With that of his brother, in 1913, his life
was almost wholly shattered, crushed, buried in the
tomb.

With the marriage of the last of his sons and his two
youngest daughters almost all the life of the house,
all the caressing grace of light, considerate footfalls,
of clear tender voices, of smiles and kisses, had
forsaken the old man, to return only in passing and at
distant intervals. His isolation became more and
more complete.

Was all over? No, this was hardly the beginning of

his afflictions. In the great silence of the harmas
there burst of a sudden [384]the terrible thunderclap of
war which roused to a protest of intolerable grief the
uttermost fibres of his being.
The whole man suffered. The Frenchman, to see his
beloved country the victim of the brutal and
underhand aggression of a predatory nation: the
father to see his dear children, a son and two sons-
in-law, cast into the furnace; the idealist and the
great-hearted man who had held war to be a relic of
barbarism, doomed to disappear from the annals of
the human race, to see war declared, and spreading
with the violence of a conflagration, surpassing in
horror all that history tells us of the armed conflicts of
the past.

Before the bloody vision of the battlefields, how

should he not feel shaken to the depths of his being
by the tremors of a terrible anger and a vast pity, he
who had never been able to see an insect suffering
without a pang at the heart?

True, in his incomparable Iliad, the Homer of the

Insects had often described creatures that hunt one
another, kill one another, devour one another with
indescribable ardour and ferocity, and he knew that
he had only written a chapter of that “struggle for life”
which is to be found on every step [385]of the biological
ladder, with the same disregard of weakness and
suffering.
But he would fain have seen man assert his
superiority over the animals by repressing these
instincts, which come from below, by the free flight of
the aspirations vouchsafed from above, by the
progressive subordination of the brute power of force
to the spiritual power of justice and love.

While these distressing problems were filling his

mind, and while, in protest against happenings so
utterly contrary to his ideas, he would thump his fist
upon his famous little table, a woman was moving
gently to and fro, playing the parts, alternately, with
the same calm countenance, of Martha and of Mary;
and when he asked her her secret, she showed him
her crucifix and read the Gospel to him, as though to
wring from his heart the cry that was uttered by the
poet of La Bonne Souffrance: 11

“Vingt siècles de bonté sont sortis de ces mystères,

Je crois en toi, Jésus.…”

In moments of affliction, Fabre is even closer to the

Truth than on the heights of knowledge and fame.
For we are never [386]nearer the God of the Gospel
than when we most feel the want of Him.
[Contents]

IV

More than ninety years of life and almost as many of

labour, nearly five years of overwhelming fame, and
almost as many of unspeakable suffering: must not a
man be “built of heart of oak,” as they say in
Aveyron, to survive so many trials?

Like the oaks of his native parts, the patriarch of

Sérignan continued to brave the assaults of time, and
even when he began to feel that his life was
declining, it seemed as though it was only
withdrawing itself from its long and manifold
ramifications in the external world to take refuge, as
in an inexpugnable asylum, in the depths and roots
of his being. He was one of those of whom people
say with us that they “cannot die.”

Fabre’s work is immortal—that is agreed. But the

artisan?

Let us resume our comparison. Like the oak that

loses its boughs, one after the other, he saw falling
one by one the several factors of his life. His life was
the harmas, that paradise of insects, that laboratory
after his own heart, where he could make his
observations under the blue sky, to the song of the
[387]Cicadæ, amid the thyme, lavender, and rosemary.

Now he was seen there no longer; hardly were the

traces of his footsteps yet visible through the
untrimmed boughs that crossed the paths and the
grass that was invading them.

His life: it was his study, his museum of natural

history, his laboratory, where, with closed doors, face
to face with Nature, he repeated, in order to perfect
them, to consign them to writing, his open-air
researches, his observations of the to-day or
yesterday. Now he no longer sets foot in it, and now
one saw—with what respect and tenderness—only
the marks left by his footsteps upon the tiled floor, as
he came and went about the big observation-table,
which occupies all the middle of the room, in pursuit
of the solution of the problems propounded by his
insects.

And we have a feeling that we are looking upon, and

handling, relics, when on this table we still see the
pocket-lenses, the microscopes and modest
apparatus which has served for his experiments. And
we have the same feeling before the collections in
the glass-topped cases of polished pine which stand
against the whitewashed walls, and before the
hundred and twenty volumes of [388]the magnificent
herbarium which stand in a row beneath them, and
before the innumerable portfolios of mycological
plates, in which vivid colour is blended so well with
delicacy of drawing, and before the registers and
stacks of notes in fine, clear handwriting, without
erasures, which promised a fresh series of
Souvenirs.

Must they be left thus abandoned previous to their

being dispersed or falling into other hands—all these
precious fragments of an incomparable life, and
these venerable premises, consecrated by such rare
memories?

The great naturalist’s disciples could not resign

themselves to the thought, and by a touching
inspiration of filial piety they have found the means to
secure these treasures, as by a love stronger than
death, against this harrowing dispersal.

To keep the dead in their last dwelling, or attract

them thither, the ancient Egyptians used to place
there the image of their earthly dwelling, offering
them at least a reduced facsimile of their life’s
environment, of the objects and premises which had
in some sort made part of their life and their soul.
Fabre’s friends sought to do still better. In order to
preserve it in its integrity, they [389]determined to
acquire the Harmas, with its plantations, its
collections, and all its dependencies, and in order to
make their homage as complete as possible they
made, with this object, an appeal for international
subscriptions, which were unhappily interrupted by
the war.

“This is the museum which we wish to dedicate to

him,” said the chief promoter of this pious
undertaking, 12 “so that in after years, when the good
sage who knew the language of the innumerable little
creatures of the country-side shall rest beneath the
cypresses of his harmas, at the foot of the
laurestinus bushes, amidst the thyme and the sage
that the bees will still rifle, all those whom he has
taught, all those whom he has charmed, may feel
that something of his soul still wanders in his garden
and animates his house.”

However, the soul of the “good sage” which they thus

sought to capture and hold here on earth—in short,
to imprison in his work and its environment—made
its escape and took flight toward loftier regions and
wider horizons.
To see him in the twilight of the dining-room [390]where
he silently finished his life, majestically leaning back
in his arm-chair, with his best shirt and old-fashioned
necktie, his eyes still bright in his emaciated face, his
lips fine and still mobile, but thin with age and at
moments trembling with emotion, or moved by a
sudden inspiration—to see him thus, would you not
say that he was still observing? Yes, but his
observations are now of an invisible world, a world
even richer in mysteries and revelations than the
world below, so patiently explored for more than fifty
years.

One day, when two professors of the Grand-

Séminaire de Saint-Paul-Trois-Châteaux 13 had come
to see him, as the time drew near to bid them good-
bye, the old man held out his hands and tucked them
under their arms, and, not without difficulty, rose from
his arm-chair, and arm-in-arm with them advanced,
tile by tile, to the threshold of the house, whither he
had determined to accompany them. Suddenly,
pressing their arms more closely and alluding to their
cassocks and their vocation, he said, energetically:
“You have chosen the better part”; and, holding them
back for a last word, he [391]added: “Life is a horrible
phantasmagoria. But it leads us to a better future.”
This future the naturalist liked to conceive in
accordance with the images familiar to his mind, as
being a more complete understanding of the great
book of which he had deciphered only a few words,
as a more perfect communion with the offices of
nature, in the incense of the perfumes “that are softly
exhaled by the carven flowers from their golden
censers,” amid the delightful symphonies in which
are mingled the voices of crickets and Cicadæ,
chaffinches and siskins, skylarks and goldfinches,
“those tiny choristers,” all singing and fluttering,
“trilling their motets to the glory of Him who gave
them voice and wings on the fifth day of Genesis.” 14

This last passage might be underlined, for now more

than ever, in our thoughts of this scientist, of whom it
has been said that “with a taste for Nature he has
given us an appreciation of God,” the work cannot be
divorced from the artisan without the grossest
inconsistency.

One who had the good fortune to become intimate

with Fabre during the last days of his life tells how
eagerly the naturalist [392]used to accept the wild
flowers which he brought in from his walks, how
tenderly he would caress them with his frail fingers
and brilliant eyes. Both looks and gestures
expressed an infinite admiration for the pure and
simple work of Nature as God has ordained it:

“And when one evening,” says his friend, “I remarked

that these little miracles clearly proved the existence
of a divine Artificer: ‘For me, I do not believe in God’
declared the scientist, repeating for the last time his
famous and paradoxical profession of faith: ‘I do not
believe in God, because I see Him in all things and
everywhere.’ ”

Another day he expressed his firm and profound

conviction to the same friend, in a slightly different
form. “God is Light!” he said dreamily.—“And you
always see Him shining?” “No,” he said suddenly,
“God does not shine; He obtrudes Himself.”

The man who thus bows before God has truly

attained, on the heights of human knowledge, what
we may call with him the threshold of eternal life. To
him God sends His angels to open the gates, that he
may enter by the straight paths of the Gospel and the
Church. [393]

After the death of Mme. Fabre in 1912, a nursing

Sister of the Congregation of Saint-Roch de Viviers
was installed at the Harmas; her name was Sister
Adrienne.
The old man appreciated her services so greatly that
he was overcome with dejection by the very thought
that she might be recalled by her superiors,
according to the rule of her Order, after the lapse of a
certain period of time. And he would gratefully press
her hand when the good Sister sought to relieve his
anxiety and inspire him with the hope that she would
be allowed to remain in his service till the end of his
days.

He found her simplicity, her delicacy, her good

nature, and her devotion so delightful that he could
not refrain from telling her so plainly in the direct,
forcible manner familiar to him: “You are invaluable,
Sister; you are admirable. I love religion as you
practise it.”

“He has often told me,” she writes, “that when he

could not sleep at night, he used to pray, to think of
God, and address to Him a prayer which he would
himself compose.”

In the spring of 1914 the aged naturalist, who was

more than ninety years of age, felt that his strength
was failing more perceptibly, [394]so that the doctors
diagnosed a fatal outcome in the near future.
On receiving the news of this alarming condition,
Monseigneur the Archbishop of Avignon hastened to
the Harmas. The invalid expressed his delight and
gratitude for the visit. Their relations were so cordial
that the prelate decided to continue them by a series
of admirable letters which have fortunately been
published.

In these letters, with great delicacy, Monseigneur

Latty avoided all that might run contrary to the
naturalist’s opinions, and very gently endeavoured to
induce him to die as a Christian.

To draw him more surely to the light that shines from

the Cross and the grace which raises the soul above
itself, he asks him to recite every evening, in unison
with him, the beautiful prayer of the dying Saviour,
which he calls “the prayer of the heights,” the height
of Golgotha, the height of life: In manus tuus Domine
commendo spiritum meum. (Into Thy hands, O Lord,
I commend my spirit.)

However, Fabre was not yet at the end of his

Calvary. Contrary to the expectation of the doctors, a
return of strength enabled him to live to see another
Spring, and it needed [395]nothing less than the terrible
shocks of the tempest unloosed upon Europe to
overcome the powers of resistance that had braved
so many storms.

During the summer of 1915 his weakness grew more

marked, so that there was no hope of many more
days of life. The curé of Sérignan having been
mobilised, the absence of the priest at this time was
a cause of great anxiety to Sister Adrienne—always
on the watch for the soul ready to escape her.

Providence happily came to her assistance; and a

Breton priest, who had come to the South to recover
his health, and had for some time been acquainted
with the master, was admitted to terms of intimacy.
After some hesitation he decided to speak to the
scientist of the Sacrament of Penitence. With that
beautiful simplicity of his, and to the astonishment of
the priest, Fabre, who seemed expecting the
invitation, replied:

“Whenever you will.”

“Purified by absolution, fortified by the Extreme

Unction, received, in full consciousness, into the
Church, Fabre displayed a wonderful serenity.
Pressing the hand of the priest who was officiating,
he listened to the recommendation of the soul. And
when he [396]heard the sacred words that were
familiar to him—In manus tuus, Domine—his lips
moved as though to pronounce the Amen of supreme
acceptance, while his gaze, which was beginning to
grow dim, settled upon the Sister’s crucifix.”

It was the 11th October 1915, at six o’clock of the

evening, that the great scientist so gently
surrendered his soul to God.

The obsequies, celebrated on the 16th October,

“were simple and affecting, as he would have liked
them to be. For a few moments before leaving the
church, the old naturalist’s fine face was again
exposed. It reflected an immense serenity. On his
peaceful features one divined the satisfaction of the
man who is departing with his work accomplished. In
his parchment-like hands he clasped a wooden
crucifix with ivory tips. Beside his head was a wreath
of laurestinus. Beside one arm was his great black
felt hat.”

The service was celebrated by the Arch-priest of

Orange, in the little church; and then the harsh, rocky
soil received the body of him who had so often
stooped over it.

This “life of J. H. Fabre told by himself” would not be

complete if we did not give here the text of the
epitaph which he himself had composed beforehand.
It is [397]magnificent: it gives one the impression of an
unfurling of wings:

“Quos periisse putamus

Præmissi sunt.
Minime finis, sed limen
Vitæ excelsioris.”

Fabre was preceded to the tomb by several months

by Mistral, who was seven years his junior. “Very
different in an equal fame, these two men are
inseparable. Mistral and Fabre both represented
Provence; one was born there and never left it, and
to some extent created it; the other adopted and was
adopted by it, and, like his illustrious compatriot,
covered it with glory.” 15

But while Fabre represented Provence, which saw

the unfolding of his rich and vital nature, and while it
lavished upon him all the beauty of its sky, all the
brilliance of its Latin soul, all the savour of its musical
and picturesque language, and all the entomological
wealth of its sunny hills, he none the less represents
the Rouergue, whence he derived his innate qualities
and his earliest habits, his love of nature and the
insects, his [398]thirst for God and the Beyond, his
indefatigable love of work, his tenacious enthusiasm
for study, his irresistible craving for solitude, the
strange, powerful, striking and picturesque grace of
his language, his almost rustic simplicity, his blunt
frankness, his proud timidity, his no less proud
independence, and with all these the ingenuous and
unusual sensitiveness and sincere modesty of his
character.

THE END

1 This chapter was written by the Abbé Fabre especially for the English edition.—
B. M. ↑
2 This was the pilgrimage of the young girls of the Université des Annales
politiques et littéraires. ↑
3 The French words are “Cousins,” “Cousines.” Cousin = cousin, good friend,
crony.—B. M. ↑
4 Jules Clarétie, Jean Richepin, Adolphe Brisson, etc. ↑
5 E. Lavisse, quoted by Dr. Legros, op. cit., p. 81. ↑
6 M. l’Abbé Germain, ex-curé of Sérignan. ↑
7 François Fabié. ↑
8 In Provence, as in Italy, the plaster statues sold by itinerant Italians are known
as santi belli = beautiful saints.—B. M. ↑
9 The text is from Ecclesiastes, i. 2: “Vanity of vanities, all is vanity,” but Fabre
cites it according to the Discours contre Eutrope, in which he had learnt it at
school, alluding to the appropriate reflection of Saint John Chrysostom: Ἀεὶ ρεν,
ράλιστα Σενπνε ἠχαιρον εἰπεῖν; ματαιότης, etc. (Semper quidem, nunc vero
maxime opportunum est dicere: Vanitas, etc.) ↑
10 Psalm 100, verse 3. ↑
11 Françoise Coppée. ↑
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