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BIOLOGICAL STAINS

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A Handbook on the Nature and Uses of the Dyes
Employed in the Biological Laboratory

By H. J. conn

Secretary, Biological Stain Commission

Prepared vnth the collaboration of

Various Members of the

Biological Stain Commission

SIXTH EDITION

Revised with the assistance of
Elmer H. Stotz and Mary A. Darrow

Published for the

biological stain commission

by
THE WILLIAMS & WILKINS COMPANY

Baltimore 2, Maryland

COPYRIGHT, 1925, 1929, 193G, 1940, 1946, 1953

Biological Stain Commission

Made in United States of America

TABLE OF CONTENTS

PAGE

Preface 5

Chapter I. Uses and standardization of biological stains 9

Chapter II. The general nature of dyes and their classification 23

Chapter III. The theory of staining 43

Chapter IV. Spectrophotometric and chromatographic analysis of dyes 53

Chapter V. Nitre and azo dyes 65

1. Nitroso dyes 65

2. Nitro dyes 66

3. Azo dyes 69

Mono-azo dyes 70

Di-azo and poly-azo dyes 85

Chapter VI. The quinone-imine dyes 101

1 . The indamins 101

2. The indophenols 102

3. The thiazins 103

4. The oxazins 117

5. The azins 125

Amino-azins or eurhodins 125

Safranins 128

Indulins 134

Chapter VII. The phenyl methane dyes 137

1. Diphenyl methane derivatives 138

2. Triphenyl methane derivatives 139

Di-amino triphenyl methanes 141

Tri-amino triphenyl methanes (rosanilins) 146

Hydroxy triphenyl methanes (rosolic acids) 171

3. Diphenyl-naphthol methane derivatives 173

Chapter VIII. The xanthene dyes 177

1. The pyronins 177

2. The rhodamines 180

3. Fluoran derivatives (eosins) 184

4. Phenolphthalein and the sulfonphthaleins 197

5. Acridine dves 206

Chapter IX. The natural dN-es 211

1 . The indigo group 211

2. Cochineal products 213

3. Orcein and litmus 216

4. Brazilin and hematoxylin 218

Chapter X. Miscellaneous dyes, pigments and histochemical reagents 225

1. Anthroquinone dyes 225

2. Thiazole dyes 230

3. Quinoline dyes 232

4. Diazonium and tetrazonium salts 232

5. Tetrazolium salts 235

6. Mineral pigments 237

Chapter XI. Neutral stains 239

74219

PREFACE

The general purpose of this book was well explained in the pref-
ace to its first edition, and that preface is, therefore, reprinted on
the following pages. The various editions which have followed
have seen a gradual enlargement of the book, with greater accuracy
of detail; but its purpose and general scope have not been changed.

A longer period (seven years) has elapsed since the fifth edition
than occurred between any other successive two. This has not
been so much because of the lack of new material on the subject as
because of difhcvdty in organizing that material in the best form
for presentation. There is a smaller increase than usual in the
number of new dves described in this edition; but this is offset bv
the addition of histochemical reagents which are not dyes in them-
selves, but develop color when properly applied to tissue containing
the compounds which the reagents are intended to demonstrate.

Although reference is given to certain histochemical reagents,
the general subject of histochemistry (in spite of its growing im-
portance) is treated very sketchily. This book hardly seems the
place to consider that field in any detail, especially considering
that new text books dealing with the subject alone and written by
specialists in the field, are now beginning to appear. Neverthe-
less, one of the important changes in this edition, in comparison
with the fifth, is in its references to histochemical usage of certain
dyes and dye-like reagents.

Perhaps the greatest change in this edition is the greater stress
laid on methods of identification of stains. All previous editions
have referred to spectrophotometric measurements and their use
in characterizing dyes; but beyond giving the absorption maxima
(sometimes not fully verified) under the individual description of the
stains, such data were largely omitted. At present, the Biological
Stain Commission has much more material on this subject avail-
able, from data that it has been collecting during the last five years
in its laboratory at the University of Rochester Medical School,
and it seems worth while to include some of that material in this
book. As a result, this book contains some 20 illustrations in
which spectral graphs of nearly all the stains now on the certifi-
cation basis are included. In every case these graphs are of typical
samples of the stain in question, selected after a comparison of
numerous batches of the same dye which have been accumulating
in the Commission laboratories for twenty years or more. These
curves are very useful to the Stain Commission in the identifi-
cation of stain samples submitted for certification^ and it is hoped
that their inclusion here may increase the familiarity of users with
the optical properties of dj^es and help them to use the stains more
intelligently.

6 rrejace

Chromatography, a still more recent method of dye identifica-
tion, is discussed in this edition for the first time. It is not treated
extensively, however; its importance is appreciated, but methods
of applying it have not yet been suiBSciently standardized to justify
its routine use in stain identification. Perhaps the brief references
to the method given in this edition will stimulate the research the
method needs to make it a more useful tool in this work.

Users of the book will also notice that there has been a slight
rearrangement of the material, with the chapter on Theory of
Staining placed earlier in the book and one additional chapter
added to include Miscellaneous Dyes, Pigments and Histochcmical
Reagents.

H. J. Conn
Geneva^ N. Y.
October, 1952

PREFACE TO FIRST EDITION

When microscopists first began, 'in the sixties and seventies, to
use stains, the demand for dyes for this purpose was naturally too
small to justify a special source of supply. They therefore had to
make use of textile dyes, which were then very crude and were
not constant in their composition. After a number of years,
however, the demand for biological stains grew and a special com-
mercial source of supply for them first appeared in Germany.
This was the Dr. G. Grubler and Co. This company did not
manufacture the dyes, as used commonly to be thought in other
countries; but on the other hand it cannot be denied that its
founder made a distinct contribution to science in making the
first effort to secure constancy and reliability in dyes intended
solely for the use of the microscopist. It is supposed that he
tested dyes under the microscope himself, and if a batch proved
satisfactory in his experience bought a supply large enough for a
number of years, bottled it under his own label and sold it to
biologists. There is no question but that in this way the biologist
was furnished with a much more reliable line of stains than if he
had been obliged to buy directly from the dye manufacturers; but
it was an empirical method of standardization and there was
nothing to prevent different batches of some dye secured by this
company from varying considerably in their composition. Such
upon investigation has proved to be the case.

Although a great service was done to biologists by this company
in the latter part of the nineteenth century, such methods of
standardization are not in keeping with modern scientific knowl-
edge. A recent cooperative undertaking has therefore been or-
ganized in America to put the standardization of stains upon a
scientific basis. This undertaking started after the war had
caused a shortage of stains, with the object of securing a reliable
supply when the foreign sources were unavailable. It has since
then been widened in its scope; and now that the foreign products
are again available, the purpose of the work is to effect a scientific
standardization of stains whether derived from foreign or domestic
sources. As a matter of fact, so far only domestic samples have
been considered. This has not been because of any prejudice
against foreign stains, but because of practical diflSculties; it is, in
brief, difiicult to test each batch before it is put on the market
when the concern handling it is in Europe.

The organization through which this work is being carried on is
known as the Commission on Standardization of Biological Stains.
It was organized in 1922 under the auspices of the National Re-
search Council and is still affiliated with it, although now no longer
a part of the larger body. It is in effect a coordinating com-

8 Preface to First Edition

mittee representing the American Chemical Society, the American
Society of Bacteriologists, the Society of American Zoologists, the
Botanical Society of America, the American Association of Pathol-
ogists and Bacteriologists and the American Association of
Anatomists. It has a membership of about sixty biologists,
members of the various societies just mentioned, who assist in the
examination and testing of stains, each in those particular lines
of technic with which he is especially familiar. It has secured the
cooperation of chemists, dye manufacturers and stain dealers, so
as to be sure that the needs of biologists can be immediately re-
flected in the supply of stains on the market. Its affairs are man-
aged by an executive committee of five members, the present
members of which represent bacteriology, botany, dye chemistry,
pathology and zoology, respectively. This executive committee
has undertaken the preparation of this book. The authorship of
the book has been assumed by the chairman of the committee,
however, in order to fix the responsibility and to make bibliogra-
phic references to it simpler than in the case of plural authorship;
but the assistance of the other committee members in the work
has been so great that they may be practically considered co-
authors of the book. The chairman of the committee, therefore,
wishes to take this occasion to acknowledge the invaluable assist-
ance given by these other members. Without their cooperation
such an undertaking would have been impossible.

The chief object of the book is to present in logical form the
information which has been accumulating in the hands of the Com-
mission since it was organized. It is neither a treatise on dye
chemistry nor one on microscopy; although it contains information
in both fields. It is an effort to present in a form acceptable to
biologists the principles of dye chemistry so far as they have a
bearing on biological stains; and to discuss the suitability of the
different dyes for various biological purposes, presenting data
partly original and partly drawn from the literature. The sub-
ject matter is realized to be incomplete, particularly that part of
it which deals with the biological uses of dyes. An effort has been
made to list the most important present uses of stains, and of the
obsolete uses to mention those of historical significance; but it is
realized that there must be many omissions. It is hoped that
readers of the book will cooperate by calling to the author's at-
tention places where the treatment of any subject seems in-
adequate.

H. J. Conn, Chairman,
Commission on Standardization
of Biological Stains.
Geneva, N. Y., 1925

CHAPTER I
USES AND STANDARDIZATION OF BIOLOGICAL STAINS

WHAT ARE STAINS?

A QUESTION frequently asked is, "what are biological stains
and how do they differ from dyes?" The answer is simply
that they are dyes, but are adapted for very special purposes.
The manufacturer thinks of two classes of dyes, those intended for
general purposes and those specially adapted for biological use.
To him the "biologicals" are a class in themselves, notable chiefly
because of the care with which they must be manufactured and
the necessity of having them agree with rigid specifications so that
they will be satisfactory for the delicate procedures in which they
are employed. To the biologist, however, there are several classes
of products included among the "biologicals" and not all of them
are stains; there are medicinal dyes, bacteriostatic agents, and in-
dicators, which are not truly stains but are put in the same group
by the manufacturer. Strictly speaking, however, a biological
stain is a dye used for making microscopic objects more clearly
visible than they would be unstained. Such a dye may also serve
for textile purposes as well; but usually a special grade is needed
for biological use. The same statement is equally true (perhaps
more so) of medicinals and indicators; and since chemically they
are all very similar they are all considered in this book. Chief
stress, however, is laid on those which are employed primarily as
stains.

THE HISTORY OF STAINING

Considering how dependent microscopists are today upon the
use of stains, it is hard to realize that much important work had
been done with the microscope before the use of stains was at-
tempted. Although natural dyes such as carmine and indigo
were well known in the early days of the microscope, their use in
staining microscopic preparations does not seem to have become
common till about 1850; and anilin dyes were not put on the
market until 1856. Yet anyone who has studied the history of
biology must realize that many discoveries had been made with
the microscope before this period.

It is safe to say, nevertheless, that the use of stains revolu-
tionized microscopic technic. The early microscopists were able
to make much progress without stains because of their painstaking
diligence. The work without stains must have been extremely
difficult, and it is hard on reading some of the old publications to
believe that some of the minute structures described were actually
seen. Few users of the microscope today would be likely to have
either the patience or the eyesight to do the work described in

9

10 Biological Stains

those early days. The fact that the microscope is now being used
successfully in the hands of so many students who would not think
of comparing themselves with the pioneers in microscopy is due
to the use of stains more than to any other factor — although, of
course, no one can deny that modern improvements in the micro-
scope have also played a part of great importance.

As a matter of fact, a revolution of a different sort, which has no
relation to staining, is in progress at the present time. The last
few years have brought out more radical changes in microscope
design — including that of the electron microscope — than have been
seen for the greater part of a century. What will be the outcome
of such radical departures as the use of electron beams, X-rays,
phase differences, reflecting optics, is still impossible to predict.
One might almost assume that the use of improved types of micro-
scopes would eventually eliminate the need of stains to render
objects more easily visible — were it not for the equally rapid de-
velopment in histochemistry, in which field dyes or similar reagents
are apt to be needed for a long time to come.

The present is hardly the place to go into the history of staining
in any detail; it has been adequately treated elsewhere. Gierke
(1884) w^as the first to discuss the subject, and presented a fairly de-
tailed account of the early uses of biological stains, as they were
understood at that time in Germany, and brought the subject up
to the date on w^hich he wrote. Conn (1933, 1948) has presented a
rather more cursory review on staining history, up to modern
times, based on a study of moderately recent publications without
making any search through old texts. Such a search has been
made recently, however, by Lewis (1942) who has presented the
most thorough review of the beginnings of staining that has yet
appeared, and shows that its origin can be traced way back to
Leeuwenhoek, the ''father of the microscope", and the first scien-
tist to see bacteria.

Histological technic, as we know it today, seems to have become
fairly well understood about 1860, and it embraced the use of cer-
tain natural dyes, notably carmine, but also indigo, madder,
saffron and phytolacca. This rapid development of the subject
followed a paper by Gerlach (1858) in which he particularly called
attention to importance of staining (with carmine) in histology.
It is not certain whether or not Gerlach thought he had originated
the idea; but it is clear that he was hailed as the father of staining
by many of his contemporaries, particularly in Germany. Care-
ful students of the subject, however, like Gierke, took pains to ex-
plain that though Gerlach had established (eingefurt) staining
technic, the actual use of dyes in microscopic work preceded him.
Gierke cites Goppert and Cohn (1849) as the earliest users of bio-
logical stains. Subsequently, however, the introduction of stains
into microscopic work has been ascribed to Ehrenberg (1838);

Uses and Standardization of Stains 11

then to Hill (1770); while now Lewis (I.e.) quotes from a letter
written by Leeuwenhoek to the Royal Society in 1714 (published
1719) in which he mentions staining sections of muscle fibers in
saffron to increase the visibility of the particles as viewed under
one of his high power lenses.

One can never be certain how long any particular individual will
remain credited with being the "first" to do something; but it is
difficult to see how the use of dyes in microscopy can precede the
invention of Leeuwenhoek's microscopes.

Following the work of the pioneers in staining the development
of the subject was rapid, particularly after hematoxylin had been
introduced by Waldeyer (1863) and more successfully by Bohmer
(1865), anilin dyes by Beneke (186i2) and alcohol differentiation
by Bcittcher (1869).

Gierke (1884), in his historical discussion of staining, says that
the history up to his day was divided into three periods, each
occupying a decade. The first decade, the fifties, was char-
acterized by a few important but unrelated discoveries which
ended in the work of Gerlach — each investigator following up
accidental observations on the staining powers of carmine and the
other well known dyes of those days. After Gerlach's work, the
development of the technic in the sixties was more rapid and de-
pended less upon chance success by the individual investigator;
the effort was made to use similarly all the dyes and metallic colors
then available. The next decade would have had much less left
to develop in this line if it had not been that by this time the great
variety of anilin dyes were available and microscopists were con-
stantly finding new uses for them. Gierke wondered if there
would be any opportunity for equal development during the ten
years to follow his paper.

That development did not stop in his day is well known.
Scarcely a year has passed without the introduction of some new
staining technic of considerable importance. Sometimes dyes
hitherto unknown to the biologist have been shown to be valuable
in bringing out some particular structure; at other times new com-
binations of dyes have proved of special value for other purposes;
while by other investigators it has been shown that old methods
used with modern refinements of apparatus and technic, may
bring out details not dreamed of by the early histologists.

USES OF STAINS

Only a brief summary of the uses of stains is given here. More
detailed citations are given under the discussion of the individual
dyes throughout this book. Certain very general texts of the sub-
ject are also useful; among those which have proved most valuable
sources of the information given in these pages are: Mann (1902),

12 Biological Stains

Ehrlich (1910), Krause (1926-7), Lee (1950, and earlier editions),
McClung (1951, and earlier editions).

Animal microtechnic. Although the earliest uses of stains were
botanical, modern histological technic was first developed on
zoological material. As a result the first extensive use of stains
was in animal histology. It is still true that there are many more
staining procedures developed in animal histology than in plant
histology, and that many more dyes are thus employed in the
former field than in the latter. (This is not true, as will be ex-
plained below, in regard to cytological methods.)

In animal histology one thinks first of the general tissue stains.
These involve the use of one, two, and occasionally three dyes in
staining sections of general animal tissue designed primarily to
differentiate nuclei from cytoplasm of cells and to permit dis-
tinctions between the various types of tissue. Best known of
these are the various procedures calling for hematoxylin, some-
times alone and sometimes with a counterstain, such as eosin Y,
Congo red, or safranin; the hematoxylin-eosin combination is in
such general use that it is commonly referred to as the "H-E
stain". Although these hematoxylin procedures have received
many modern refinements, they date back to the 19th century and
still bear the names of Heidenhain, Delafield, and Mayer, men
who did their chief work in the eighties and nineties. Also in-
cluded among the general tissue stains are various combinations
of basic dyes such as crystal violet, methylene blue or one of the
azures, with some contrasting acid dye such as eosin Y; the former
to stain the nuclei, the latter, the cytoplasm of the cells.

Somewhat more specialized in their application are the con-
nective tissue stains. The distinction between them and the general
tissue stains is more or less arbitrary, particularly because some
procedures that bring out connective tissue and elastin to good ad-
vantage are also fine general tissue stains. The grouping can,
however, be made in a rough way and for practical purposes is
rather convenient. Of special note among the connective tissue
stains is a triple-staining procedure proposed by Mallory, which in
its original form called for the dyes, anilin blue, orange G, and
acid fuchsin; its important feature is mordanting in phosphotungs-
tic acid before applying the final staining fluid. It has been
variously modified in recent years and the variants are usually
called modifications of the "Mallory anilin blue connective tissue
stain", although some of them omit the anilin blue entirely.
Among connective tissue stains are also included a variety of
double, triple, and even quadruple stains of a miscellaneous nature,
and the well-known Mallory phosphotungstic acid hematoxylin
method, in wdiich such strong polychrome properties of the dye
are developed that no counterstain is necessary to secure proper
differentiation of tissue.

Uses and Standardization of Stains 13

A third important group of methods in animal microtechnic in-
cludes those for neurological staining. These methods are very
numerous and all highly specialized. Many of them (as is true of
some methods for demonstrating connective tissue) are not actually
dyeing procedures, but depend on the use of silver salts with which
tissue elements are impregnated so that the desired structures are
made visible when the salts are converted into metallic silver. As
these silver methods do not involve dyes, no attention is given to
them in the following pages. (They are, however, described in
Staining Procedures,* leaflets I-B and I-C.) This is not to say,
however, that dyes are not used by neurologists in microtechnic;
many such methods, as a matter of fact, do call for dyes, either with
or without impregnation of the tissue with metallic salts.

In addition to these three groups of procedures in animal his-
tology, there are stains for other types of tissue and body fluids.
Fats, for example, are stained by special oil soluble dyes, such as
Sudan IV. Blood has its own special methods of staining, most of
which depend on the use of compound stains, prepared by allowing
eosin or a related dye to react upon a mixture of dyes of the meth-
ylene blue group; such compound dyes are also useful in staining
bone marrow. Bone has its own special methods, many of which
depend on the use of alizarin red S, which is particularly valuable
for demonstrating bone in small specimens that have been cleared
by treatment in klkali.

Histochemistry. Many of the above-mentioned specialized
staining methods come close to yielding actual chemical informa-
tion as to the nature of cell constituents. Some theories of stain-
ing do postulate chemical affinities to explain differential staining;
but they do not go very far in the matter, chiefly because stains
are ordinarily applied to fixed tissue, whose chemical nature is
realized to be very different from that which occurs in nature.
The introduction, however, of rapid freezing methods is now en-
abling histochemists to get away from chemical fixation and is
giving them material for study which is more nearly representative
of natural conditions. Also their study of enzymes on the one
hand, and thejir identification of the individual nucleic acids on the
other are gradually giving them criteria to show which stains or
other reagents are actually specific for definite cytochemical in-
gredients. Especially important has become the use of the Schiff
reagent (fuchsin-sulphurous acid), after some oxidizing agent, in
the identification of mucopolysaccharides. Developments in histo-
chemistry are now becoming so rapid that predictions are hard to
make. Whether the histochemical reagents of the future will be
primarily dyes or other chemicals, it is impossible to say at present.

Plant Histology. A rough grouping of the most common plant
histological methods can be made by recognizing general tissue

*Conn and Darrow (1943-52).

14 Biological Stain.s

stains and stains for woody tissue. This grouping is no more
absolute, however, than the separation between general tissue
stains and connective tissue stains in the zoological field. Prob-
ably the most frequently used dye in botanical microtechnic is
hematoxylin, which is employed in a number of procedures either
for general tissue or woody tissue. Another very valuable dye is
safranin which is one of the best nuclear stains employed in
botanical work; with a green or blue counterstain, very effective
pictures are obtained.

Various combinations of stains have been employed for certain
more highly specialized purposes, such as staining fungi in tissues,
or for demonstrating pollen tubes in the style. For such purposes
as this, numerous dyes have been called for, such as thionin,
orange G, martins yellow, malachite green, acid fuchsin, methyl
gjeen, carmine, basic fuchsin, and lacmoid. It is nevertheless
true, as stated above, that there is by no means such a variety of
staining procedures in plant histology as in the zoological field.

Plant Cytology. Although the statement just made is true in
the general histological field, it cannot be made in regard to cytol-
ogy. The earliest cytological work was done with plant material,
probably because of the greater ease with which rapidly dividing
cells (meristematic tissue) can be obtained from plants than from
animals. The result is a very considerable variety of procedures
in plant cytology. Best known among them, undoubtedly, are:
the variations of the hematoxylin methods; the Flemming triple
stain, which calls for crystal violet, safranin, and orange G; and the
aceto-carmine method employed for fresh smears of anthers. There
are numerous special combinations of crystal violet and of safra-
nin, each with particular counterstains designed to bring out certain
desired effects.

It is interesting to note that, although there are many dyes used
in both plant and animal microtechnic, there are others that are
important in one of these fields but not in the other. Thus car-
mine and safranin are pre-eminently botanical stains, methylene
blue a very important zoological stain that is hardly ever em-
ployed by the botanist.

Microbiology. Another very important field in which biological
stains are employed is the study of bacteria and related micro-
organisms. The dyes thus used are somewhat limited in number,
methylene blue, crystal violet and basic fuchsin satisfying most of
the bacteriologists' needs. It is very interesting, however, to
note that these three dyes, which are pre-eminently bacteriological
stains, are by far the largest sellers of any of the biologicals. This
indicates that although the bacteriologist does not employ as
many staining procedures as the worker in other fields, he is by far
the most important customer, in point of quantity ordered, of any
to whom the manufacturer supplies these products.

Uses and Standardization of Stains 15

A comparatively new field is now bacterial cytology. As in the
case of histochemistry, development in this field has been made
possible by improved methods of fixation. The conventional
method of rendering bacterial cells stainable was merely to dry
them on a slide, a process which distorts the cell and makes inter-
nal structure quite diflficult to demonstrate. Such crude fixation
methods are now being replaced, for all delicate work, with others
that cause less distortion ; and gradually bacterial cytology is being
revealed. There are difficulties still to be surmounted; artefacts
and true nuclear structures are sometimes difficult to tell apart,
and misinterpretations are frequently made. Nevertheless, the
field is being rapidly explored, and the subject of bacterial cytol-
ogy is no longer so highly speculative as it was a comparatively
few years ago.

Bacteriological staining methods (outside of cytology) can be
divided into two groups: stains for bacteria in dried films; and
stains for bacteria in tissue. The former consist of very simple
procedures, ordinarily; about the most complicated of them is the
Gram stain which calls for crystal violet and a counterstain; some
species of bacteria taking the violet stain, others, the counterstain.
The stains for bacteria in tissue are essentially histological methods,
many of them very similar to those used for general animal tissue;
their main purpose, however, is not the differentiation between
different tissue elements but that between bacteria and the tissue
in which they are located.

An important aspect of bacterial staining is the laboratory
diagnosis of disease. In numerous diseases, of which tuberculosis
and diphtheria are the most conspicuous examples, staining pro-
cedures play a prominent role in diagnosis. So important is this
use that in spite of the smaller number of staining methods em-
ployed by the bacteriologist, he orders stains by the kilo while the
zoologist may content himself with 10-gram bottles. This is the
real reason why the bacteriologist is the chief customer of the
biological stain company.

BIOLOGICAL STAINS IN TIME OF WAR

The last mentioned use of stains has made them a very im-
portant war commodity. The first World War proved this. Be-
fore that war practically all dyes had come from Germany, and no
biological stains were considered reliable unless they bore the label
of one particular German company. In 1914, the blockade of
Germany prevented its dyes from going overseas and biologists
in many other countries began to wonder where they would get
fresh supplies of stains when the stocks of German dyes on hand
at the beginning of hostilities were exhausted. In America this
did not prove an immediate problem, for those stocks were quite
large, partly in the hands of the users and partly in the store-

16 Biological Stains

rooms of dealers, and if the demand had been only that of peace-
time, probably the situation would not have become acute for
several years. As soon as this country became involved in the
war, however, the demand greatly increased. Every Army or
Navy hospital had to have its laboratory; and because of the higher
degree of medical care given the men than in any previous war,
such hospitals and laboratories soon became quite numerous. The
demand for stains, therefore, broke all previous precedents and the
stock of German dyes available was no longer anywhere near
enough for the demand. Laboratory technicians soon were forced
to get familiar, with American dyes — and they learned, in the hard
way, that biological stains need standardization.

After that war the demand for stains seems to have decreased,
although no actual figures are available. Then, gradually, during
the next 20 years, the demand increased as new laboratories were
started and new uses for stains proposed, but it did not reach a
very high figure until 1939. Then almost simultaneously with the
German invasion of Poland, the first marked increase in the de-
mand (since the previous war) was noted. Just why the increase
occurred then in America is hard to figure; but it is possible some
users of dyes became panicky for fear that the coming of war would
cause shortages in this field. No such shortage did develop, in
America, at least; and it is well that it did not, for as soon as the
U. S. Government, through Army, Navy, and Lend-Lease agencies,
began ordering stains on the war basis, the increase in orders
exceeded anything that had been previously thought possible.
By 1943 the demand for stains was fully ten times that of 1939.
It seems almost incredible to believe that any Government in war
should find it necessary to order any dye (even such a dye as
crystal violet) by the thousands of bottles; and the fact that such
orders were received again and again gives some idea as to the
number of field hospitals called for in modern warfare.

COMMERCIAL SOURCES OF STAINS

The early biologists naturally obtained their dyes from con-
cerns whose primary business was supplying such products to the
textile industry. This proved unsatisfactory because of the great
variation in the dyes thus obtained, even though sold under the
same name. To improve the situation. Dr. Weigert, in 1880,
advised a Dr. Georg Griibler, a student of Ludwig and Dreschel
at Leipzig, to concern himself with marketing anilin dyes especially
to use in microscopy. Dr. Griibler, accordingly, founded a com-
pany (first registered as "Physiol. -chemisches Laboratorium, Dr.
Georg Griibler"; subsequently known as "Dr. G. Griibler & Com-
pany") which originally dealt with stains and other physiologico-
chemical preparations, but later specialized almost entirely in dyes.

Uses and Standardization of Stains 17

In 1896 Dr. Grlibler had to withdraw from this firm and a former partner of Dr.
Griibler's named Schmid, became the proprietor. After retiring from this business.
Dr. Grubler started a laboratory of his own which continued for some time to supply
Dr. G. Grubler & Company with certain products. This laboratory was subse-
quently incorporated in 1897 under the name of "Dr. G. Griibler's mikroskopisch-,
chemisch-bacteriologisches Laboratorium," and was sold to Dr. HoUborn. The
company thus founded subsequently became "Dr. K. HoUborn & Sohne", and for a
number of years continued its relations to the Grubler company. It did not, how-
ever, supply the latter company after 1921, but began marketing such products on
its own account.

This matter is gone into in some detail because from 1921 till about 1945 there
were two rival firms producing stains in Germany, the Dr. G. Grubler & Company
and K. HoUborn & Sons; and the relationship between these two concerns is not
generally understood. Each company made certain claims that are often inter-
preted as implying that it produced the only genuine Grubler stains. As a matter
of fact, neither company manufactured dyes and the actual claims of the two com-
panies were these: G. Grubler & Company claimed that they alone had the list of
commercial sources from which Dr. Grubler obtained his dyes some 40 years previ-
ously and the standards set up by him at that time; K. HoUborn & Sons claimed
that they were the only concern to produce the so-called "original Grubler prepara-
tions", these preparations being certain staining fluids and combinations of stains
(e.g., the Giemsa stain) first developed in Dr. Griibler's private laboratory. It will
be seen that these claims thus stated were not contradictory, but misunderstanding
sometimes resulted from the advertisements published by the two companies.

The Grubler stains were pretty generally used throughout the
world until about 1916-18. The first World War changed the
situation, because the temporary unavailability of German stains
forced England and the United States to develop their own sources
of stains. The second World War had an even greater effect on
the situation; and some time either during the war or just after,
the two above-mentioned German concerns seem to have gone out
of business. There are still German stains produced, notably by
E. Merck and Co., and the Bayer Co., and they are sold at prices
with which American stains cannot compete in the world market;
but they hardly appear on the American market now and are ex-
cluded from that of Great Britain, both of which countries now
have well established stain producers of their own.

It required some time after the first World War before the
biological stain production in other countries besides Germany
could acquire the prestige formerly belonging to the productions of
other countries. At first perhaps, there was some reason for the
unfavorable reputation attained by non-German stains. It natur-
ally required some time for the concerns just then entering the
field to learn to produce stains capable of giving the same results as
those obtained by the German product. As soon as the companies
had solved these problems, however, their products in general
were superior to those available before the war. In certain
quarters the prestige of the German stains still worked to the dis-
credit of the products of other countries; but before the second
World War began, the resulting prejudice had just about come to
an end. Although this prejudice exists no longer, special atten-

18 Biological Stains

tion must be given here to the situation which existed in the United
States and Canada, following the First World War, as it was this
which led to the establishment of the Biological Stain Commission,
the sponsor of this book.

MODERN STANDARDIZATION OF STAINS

The Biological Stain Commission is concerned with the inspec-
tion and standardization of stains, not with their manufacture, as is
sometimes supposed. It was learned in 1920, while the post-war
embargo on dyes was still in effect, that American scientists were
being supplied with dyes from three or four different stain com-
panies and that their products were not sufficiently uniform to be
reliable. Accordingly, through the cooperation of the National
Research Council of the United States and three or four American
scientific societies, the Commission on Standardization of Biological
Stains (now known as the Biological Stain Commission) was estab-
lished. The Commission is now an independent non-profit cor-
poration, having representatives on its membership of eight
American scientific societies with which it cooperates. The work
of the Commission is two-fold. First, by cooperation of various
biologists and chemists it gathers information concerning the
nature of dyes as related to their use in microscopic technic; sec-
ondly, by working with the manufacturers and dealers it en-
deavors to see that the supply of stains available in America is of
the highest possible quality as judged by their performance in
actual laboratory use. The first of these purposes has inspired
this book, which is now in its sixth edition, and at the same time
has led to the publication by the Commission of a journal. Stain
Technology and a loose-leaf manual, Staining Procedures
(Conn and Darrow, 1943-4). The second object is being brought
about by the plan of certifying stains.

The certification plan has been adopted because of the great
difficulty of drawing up any chemical or physical standards to
determine which stains are satisfactory and which are not. If
such standards could be formulated, it would be possible to draw
up specifications with which manufacturers of stains would be
expected to comply.

Specifications. Drawing up specifications for biological stains
has proved difficult. In many cases their chemistry is not en-
tirely understood, and even when it is, their behavior in actual use
is often dependent upon the amount and nature of impurities that
may be present in only minute quantities. Since these im-
purities are sometimes beneficial and sometimes detrimental and
their nature is often unknown, practical specifications are much
more difficult to draw up than in the case of ordinary chemicals
where a high degree of purity can be obtained and any impurity is
undesirable.

Uses and Standardization of Stains 19

Nearly 30 years ago the Stain Commission began trying to draw
up specifications for some of the most commonly used stains.
These specifications were published in the first edition of this
book. They did not prove very useful, however, and of recent
years no great attention has been paid to them. Much more
workable specifications have been secured during the last five
years with the cooperation of the National Formulary Committee
of the American Pharmaceutical Association.

In several recent editions of the National Formulary, published
by the American Pharmaceutical Association, a section has been
included in which formulae of staining solutions are given. Origi-
nally there was no agreement between these formulae and the
ones recommended by the Stain Commission. Beginning in 1937,
however, it was decided that the National Formulary Committee
and the Commission on Standardization of Biological Stains should
cooperate in this matter. As a result of this cooperation there
have been several worth-while accomplishments :

1. Specifications of the most important stains now on the certi-
fication basis have been drawn up and have been published in
recent editions of the National Formulary. These specifications
are partly chemical and spectrophotometric, but also contain de-
tailed statements as to how the stains should be tested as to their
behavior for biological purposes and state the results to be expected
from these tests. In every case these specifications have been
made to harmonize with the tests as actually performed by the
Stain Commission.

2. The formulae of staining solutions given in the National
Formulary, in Staining Procedures, and in the Manual of
Methods for Pure Culture Study of Bacteria, published by the
Society of American Bacteriologists, have been compared and
studied critically with the object of publishing identical formulae
in all three places. This has resulted in a few charges from the
formulae given in early editions of this book.

3. Another result of this cooperation has been to give the work
of the Stain Commission more of an official standing than it had be-
fore. The Stain Commission was originally formed as an entirely
unofficial organization and has never had any connection with any
government organization. The specifications of the United States
Pharmacopoeia and the National Formulary, however, do have
official recognition. The publication of the seventh edition of the
Formulary, therefore, with its references to stains certified by the
Commission, has resulted in their official adoption for many pur-
poses throughout the United States. Most notable has been the
adoption by the United States Armed Services in connection with
purchases made by their procurement agencies. Inasmuch as the
seventh edition of the National Formulary was published at about

20 Biological Stains

the time the United States entered the second World War, this
proved a development of considerable significance.

4. It has been realized for some time that the specifications
would be more satisfactory if they could eliminate statements that
the dye under consideration should be satisfactory for such and
such procedures. This has proved difficult because of lack of cor-
relation between chemical and optical characteristics, on the one
hand, and performance on the other. Of recent years apprecia-
tion has been growing of the fact that this lack of correlation
might be due to insufficient data; and that the only way to find
out would be to collect the data. Accordingly the assay labora-
tory of the Stain Commission has been going over its collection of
stain samples, some satisfactory and some unsatisfactory, to see if
any agreement between the two types of specification can be found.
It proves that such agreement is lacking in so many cases that
specifications based on performance must still be included; but the
data that are being collected are nevertheless proving distinctlj''
valuable. They are showing better than ever before just what
limits of variation in chemical and physical specifications are per-
missible; and as a result it is now possible to establish better defi-
nitions of and specifications for biological stains than ever before.
Much of this new information is being incorporated into this edi-
tion of Biological Stains.

Standardization Procedures now in Operation. This inter-
relation between the National Formulary specifications and the
standardization methods of the Stain Commission makes these
methods of considerable interest. It must be explained that the
standardization procedures adopted by the Stain Commission
have been entirely on the batch basis — that is, approval is not
given to the entire product of any one company, but is extended
to each individual batch that is put on the market. The reason
for this is that the Commission has never felt willing to place re-
liance on chemical or optical tests. All the specifications that
have been drawn up by the Stain Commission or by the National
Formulary Committee have contained statements which mean
essentially as follows: "The sample must prove satisfactory when

tested by the following procedures: ". It is obvious that

until it is known just how the results obtained in practical use
correlate with chemical or optical properties, any tests depending
upon performance must be carried out for each individual batch.

Commission Certified stains are- now coming to be designated
with the initials "C.C." following the name of the dye.

There is no need of listing here the tests actually employed by
the Commission; they are given in an appendix of this book where
they can be consulted by anyone who is interested. It must,
however, be explained that these tests fall into two groups : chemi-
cal and optical on the one hand; tests for performance on the other.

Uses and Standardization of Stains 21

In interpreting results, greatest weight is given to the tests for
performance. It not infrequently happens that a batch of some
stain will prove to give perfectly satisfactory results in all the
latter, but proves slightly low in dye content, or disagrees in some
respect with optical characteristics called for in the specifications.
The rule in the case of such a sample is to approve it on the basis
of its performance in spite of the slight non-conformitv in the case
of chemical or optical characteristics.

Batches of dyes thus approved by the Commission are sold by
the companies with a special label furnished by the Commission,
known as the certification label. This label indicates the certifica-
tion number of the batch and is not supposed to be employed for
any batch other than that on which it is issued. A certification label
on any bottle of stain means therefore, five things: (1) a sample of
the batch bearing the label has been submitted to the Commission
for testing and a portion of the sample is permanently on file; (2)
the sample proves true to type, as judged by spectrophotometric
tests; (3) its dye content is up to specification and is correctly in-
dicated on the label; (4) it has been tested by experts in the pro-
cedures named on the label and has been found satisfactory by
them; and lastly, (5) no other batch can be sold under the same
certification number except by such a flagrant breach of confidence
on the part of the company as to risk losing the good will of the
Commission.

At the present time (1952), 51 stains have been put on the certi-
fication basis. These include all the most commonly used
biological stains as well as a few in which the Stain Commission
lias become interested, although they are not so extensively em-
ployed in the laboratory as most of the others on the list.

There are, at the time this sixth edition goes to press, nine com-
panies in the United States and one in Canada submitting their
stains to the Commission for certification before putting them on
tlie market. It must be realized, however, that no one of these
concerns necessarily manufactures all the stains which it thus
submits; but in the case of any stain which is manufactured else-
where, the company takes responsibility for its performance as a
biological stain on the basis of tests made to show its adequacy,
and in many instances carries out a certain degree of purification
or other processing before putting the stain on the market. One
of these companies puts on the market every stain now on the
certification list. Three other companies submit samples of over
half the stains thus listed, while the other companies merely re-
quest certification of one or two dyes in which they specialize.
No dyes have yet been certified by the Stain Commission sub-
mitted by any concern outside North America. The reason for
this is because of the difficulty in handling the certification of
stains on the batch basis with a concern that is located at a dis-

22 Biological Stains

tance, doing business in this country only through agents who are
not in direct touch with the actual manufacturers of the dyes.

Although this phase of the work of the Stain Commission is one
of inspection, it has not brought about unpleasant relations with
any manufacturer or dealer in stains. As a matter of fact, the
hearty cooperation of the American stain companies has been ob-
tained throughout; and without this cooperation much that has
been accomplished in the way of standardization would have been
impossible.

The British Biological Stains Commission. Late in 1949 an or-
ganization of this name was established in England with objects
similar to those of the American Commission. The situation in
England is different from that in the United States, and has to be
handled differently. Foreign dyes have been definitely excluded
from the country for some time, and a few English concerns are
producing biological stains, without much control of their products.
It is still too soon to tell what the results will be of the English
effort to standardize their supply. They are cooperating with the
Biological Stain Commission in America, and samples of British
stains have been sent to this country for testing in the Commis-
sion's laboratories. Many of the British samples submitted do
not conform to the standards adopted in the U. S.; but there is
little doubt but that the quality of the products will be improved
now that attention is being given to the matter by a group of
scientists who use them.

CHAPTER II

THE GENERAL NATURE OF DYES AND
THEIR CLASSIFICATION

DYES are generally classed in two groups, the natural and the
artificial. The former class is now of relatively smaller im-
portance from the standpoint of the manufacturer and the
textile dyer; for the artificial dyes far outnumber them and the
advancement of science is gradually making it possible to produce
many of the formerly natural dyes by artificial means. It just
happens that one or two natural dyes, the derivatives of cochineal
and logwood extract (see Chap. IX), are among the most valuable
biological stains; but the natural dyes in general are so few in num-
ber than they can be practically disregarded in considering the
general chemical nature of dyes.

Because the first artificial dyes were produced from anilin, all of
this class are often called "anilin dyes," although there are now a
large number of them which bear no relation to this compound and
are not derived from it. Therefore the term is now quite largely
being replaced by the more correct expression '*coal-tar dyes,"
since all of them are made by chemical transformations from one
or more substances found in coal-tar.

Comparatively little is known concerning the chemistry of the
natural dyes; but the synthetic dyes have been carefully studied
and volumes have been written concerning their chemical compo-
sition and its relation to their behavior. Much of this may w^ell be
ignored by the biologist who employs dyes merely for staining
microscopic objects. A certain familiarity with the general prin-
ciples is useful, however, in helping him to employ stains scien-
tifically. This is particularly true now that there is a growing
tendency to use dyes in biological w^ork as histochemical reagents.
The brief discussion that follows is intended to help the biologist
to understand merely the fundamental principles of dye chemistry.

BENZENE

All coal-tar dyes are organic compounds of the aromatic series.
In other words they may be considered as derivatives of the hydro-
carbon, benzene, CeHe, the formula for which is usually considered
to be

CH
lie ^CH

HC /CH
\^

and is usually written in abbreviated form

24

Biological Stains

The double bonds in benzene are believed not to be fixed but to
oscillate between adjacent carbon atoms.

The importance of benzene in organic chemistry is because of
the infinite number of ways in which it can combine with other
radicals and elements, forming compounds of extreme complexity.
If the elements in such compounds are combined in certain ways
the substance is colored, and such colored compounds, after slight
additional changes in the molecule, become dyes. In this respect
the colored benzene derivatives are unlike the colored pigments of
simpler composition; many of the latter may show intense color,
but do not act as dyes.

One type of substitution in the benzene ring is specially impor-
tant in dye chemistry. Two atoms or groups having two' valency
bonds instead of one may also replace two hydrogen atoms, pro-
vided the replacement takes place simultaneously and the hydro-
gen atoms replaced are situated either in the ortho or in the para
position to each other. Thus two oxygen atoms (which are bi-
valent) may replace two hydrogen atoms (which are monovalent)
forming the compound known as quinone 0611400, the formula for
which is

o

It

/\

HC CH

I

11
O

or as commonly written

In printed formulae, such as those that follow in this book, the
quinone ring is often abbreviated still further by omitting the
double bonds within the ring. The substituent atoms or groups
may or may not be alike, so long as both have two valency bonds
entering into the combination. This type of substitution involves
a rearranging of the double valency bonds in the benzene ring; and
in compounds of this type, called quinoid compounds, the double
bonds are supposed to be fixed, not mobile as in benzene. This

General Nature of Dyes 25

change of the valency bonds takes place very readily in many
dyes, and certain peculiarities of their behavior are explained by
it; (see for example p. 199).

Three mono-substitution products of benzene are of importance
in considering the structure of dyes, namely; toluene or methyl-
benzene, CeHsCHs; phenol, carbolic acid or phenylic acidjCeHs OH;
and anilin or phenyl amine, CeHs NH2. Their constitutional for-
mulae are as follows:

CH3 OH NH2

I I I

toluene phenol anilin

Two important di-substitution products are xylene or dimethyl-
benzene C6H4 (CH3)2, and toluidine, C6H4CH3NH2. Both of
these occur in the above mentioned three isomeric forms, as shown
below for xylene:

CH3 CH3 ^H3

I CH, I I

CH3 1

CH3

ortho-xylene meta-xylene para-xylene

CH3

I NH.

ortho-toluidine

RELATION OF MOLECULAR STRUCTURE TO COLOR

Although there is still much to learn as to the explanation of
color in terms of structural formulae, it is now well known that
certain definite atomic groupings (known as chromophores) are
associated with color. The basic groupings involved in these
chromophores are C:C, C:0, C:S, C:N, N:N, N:0, and NO2; and
the more of these that occur in the same compound the more pro-
nounced the color. It will be noticed that the quinone ring, as
pictured above, contains the following chromophore grouping in
duplicate :C-C:C-C: . Quinone, itself, is in fact colored, and
the quinone ring is one of the most important chromophores
known, all compounds containing it showing intense color.

Probably if the whole situation were understood, physical chem-
istry could give us an explanation of why some compounds are

26 Biological Stains

colored. We know that color is caused by selective absorption
of certain wave lengths of light, so that the light transmitted by
or reflected from a given substance lacks these particular wave
lengths of the visible spectrum and accordingly appears colored.
Such a substance, in other words, acts as if it were a prism pro-
vided with a screen to block off certain parts of the spectrum in
which the refracted light is normally transmitted. Conceivably
the chromophores confer upon the molecule properties of just this
sort, acting possibly as a system of resonators tuned to vibrate at
the same rate as the waves to be absorbed. The theory cannot be
discussed here in any detail; but it should be pointed out that
valence electrons under certain circumstances confer resonance
on a given compound. The unsaturated compounds, such as
those containing the double bonds of the chromophores, show a
special tendency toward resonance; and it is a fact that as the
general degree of unsaturation and complexity of a compound in-
creases, the spectral absorption tends to pass from ultraviolet
toward infra-red, that is, the color transmitted (or reflected)
changes from yellow through reds and blues.

The benzene compounds containing chromophore radicals are
known as chromogens. A chromogen, however, although it is
colored, is not a dye, in that it possesses no aflSnity for fibers or
tissues. It may coat them, but only mechanically, and it will be
easily removed by mechanical processes. That is, it will not
"take". (See, however, the discussion of fat stains, p. 69). In
order for a substance to be a dye, it must contain in addition to the
chromophore group, a group which imparts to the compound the
property of electrolytic dissociation. Such auxiliary groups are
known as auxochromes. They may slightly alter the shade of the
dye, but are not the cause of the color. Their function is to fur-
nish salt-forming properties to the compound. Certain chromo-
phoric groups have also slight auxochromatic properties.

To illustrate these different types of groups, let us take a typical
example. The nitro group (-NO2) is a chromophore. When three
of these groups displace three hydrogen atoms in a benzene mole-
cule, we have the compound trinitrobenzene,

O2N NO,

NO2

which is yellow. It is not a dye, however, but is a chromogen. It
is insoluble in water, and is neither an acid nor a base; that is, it
does not dissociate electrolytically and consequently cannot form
salts with either alkalies or acids. If, however, one more hydrogen
atom is replaced, this time with the hydroxyl group (-0H), which
is an auxochrome, the resulting compound,

General Nature of Dyes 27

OH

O2N I NO2

NO2

is an acid, capable of electrolytic dissociation and of forming salts
with alkalies. It is the familiar substance picric acid, and is a
yellow dye.

It will thus be seen that the color of picric acid is due to the
chromophoric nitro groups, and that its dyeing properties are due
to the auxochromic hydroxyl group. If the nitro groups be re-
duced to amino groups (-NH2), which are not chromophores, the
resulting compound is colorless and hence is not a dye.

Summing up, we arrive at the definition of a dye as an organic
compound which contains chromophoric and auxochromic groups
attached to benzene rings, the color being attributable to the chro-
mophores and the dyeing property to the salt-forming auxochromes.

ACID AND BASIC DYES

There is frequent misunderstanding among biologists as to the
meaning of the terms "acid dyes" and "basic dyes". It is some-
times assumed that the terms refer to the H-ion concentration of
the dye solutions. Nothing could be further from the truth. The
distinction actually depends on whether the significant part of the
dye is anionic or cationic, and bears no direct relation to the reac-
tion of any solution of the dye. As a matter of fact, some of the
most strongly anionic or acid dyes, like eosin Y, regularly form
salts with strong metallic ions such as sodium; and the greater
strength of the metallic cation determines the reaction of the solu-
tion, i.e. the solution is ordinarily basic in reaction.

As the auxochromes are the salt forming groups of dyes, it is
ordinarily the nature of the auxochrome present that determines
in which of these two classes a dye belongs.

Some auxochromes are basic, e.g., the amino group (-NH2),
while others are acidic, e.g., the carbonyl group (-COOH). The
amino group owes its basic character (which it transmits to the
whole molecule) to the ability of its nitrogen atom to become pen-
tavalent by the addition of the elements of water (or of an acid),
just as in the case of ammonia; thus:

H H H H H. H H

H— N + =

\ /
H H

= H— N

/ \
H OH

and

H N +H CI =

\
H

= H— N

/ \
H CI

ammonia water

ammonium
hydroxide

ammonia hydro-
chloric
acid^gas

ammonium
chloride

28

Biological Staijis

H H

/ \
-N + =

\ /
H H

H H

\ /
■- R— N and

/ \
H OH

H

/
R— N

\
H

H H

\ /
+H— CI = R— N

/ \
H CI

amine water

hypothetical
organic am-
monium base

amine

hydro- amine
chloric hydro-
acid gas chloride

In other words, the NH2-group yields hydroxyl ions and enables
the compound to ionize and to act as a cation in forming salts;
such a dye is a basic dye.

The carboxyl group (-COOH), on the other hand, is acidic, as it
can furnish hydrogen ions by electolytic dissociation. Many of
the most important acid dyes contain this group; as they ordinarily
occur as sodium salts, the characteristic group in these dyes is
-COONa. Other acid dyes contain the rather weaker auxochrome
group -OH, and form salts in -ONa.

The more of one of these groups in a compound, the stronger
base or acid it becomes. If there is one of each, the basic character
of the amino group predominates, but is weakened by the influence
of the acidic group. The strength of both groups is also influ-
enced by other groups or atoms in the compound; thus, for ex-
ample, the chromophore -NO2, although incapable in itself of con-
ferring acid properties to the compound, exerts an influence to make
any hydroxyl group in the compound more strongly acidic, in other
words to become more highly dissociated electrolytically.

One other group of atoms encountered in dye chemistry needs
explanation, namely the sulfonic group, -SO3H. It is a salt-
forming group of strongly acidic character, in that it suffers ex-
tensive electrolytic dissociation. This group, however, is only
very feebly auxochromic. Its function is to render a dye soluble in
water, or to change an otherwise basic dye into an acidic one, as in
the case of the fuchsins, where the strongly basic "fuchsins" are
changed into the strongly acid "acid fuchsins" merely by the intro-
duction of sulfonic groups into the former. A compound which
contains a chromophore group and a sulfonic group is not a dye,
however, unless there is also present a true auxochrome group.

From this it can be understood what is meant by calling dyes
either basic or acidic. It neither indicates reaction nor does it
mean that the dyes of commerce are actually bases or acids. They
are ordinarily salts. An acid dye is a salt of a color acid — usually
its sodium salt, but occasionally a salt of potassium, calcium or
ammonium. A basic dye is a salt of a color base — usually a
chloride, but sometimes a sulfate or acetate. (The oil-soluble
dyes present an exception to this statement; see p. 76.) The terms
anionic and cationic dyes would be more suitable, but they have
never come into general usage. Basic dyes are sometimes avail-
able as free bases, in which case the name is ordinarily followed by

General Nature of Dyes 29

the word "base". Thus "basic fuchsin" means the salt of fuchsin
with some colorless acid (e.g., hydrochloric) while "fuchsin base"
indicates the color base of this dye, not combined with any acid.

THE CHROMOPHORES

As stated above, every dye contains at least one group of atoms
known as a chromophore, which is regarded as being responsible
for the colored properties of the compounds in which it occurs.
Some of these chromophores have a basic character, others acid.
There are only a comparatively small number of them which enter
into the usual biological stains, and only these need be considered
here. They are as follows :

BASIC CHROMOPHORES

1. The azo group, — N = N — , which is found in all azo dyes, of
which methyl oraiige and Bismarck brown are well known ex-
amples. In all these dyes, a benzene ring is attached to each
nitrogen atom. All the dyes of this group may be looked upon as
derivatives of azobenzene,

N = N

2. The azin groujp^

N
/

\

N

/

which is found in phenazins, of which neutral red and the safranins
are good representatives. The skeleton formula of a safranin is:

H2N N NH

in which x represents the negative ion of a monobasic acid such as
hydrochloric, acetic, nitric or sulfuric. This chromophore is
capable of a variety of rearrangements of its valency bonds, as the
bond between the two nitrogen atoms may disappear and the com-

30 Biological Stains

pound assume a quinoid structure, as for example the following
grouping :

H

H3N_/\_N_/\^NH
II LI

/ \
\ X

3. The indamin group, — N = , as observed in the indamins,
thiazins, and so forth. Methylene blue is the best known repre-
sentative of this group. In these dyes, two benzene rings are at-
tached to the nitrogen atom, one of these being in the quinoid form
and hence adding a second chromophore. The typical indamin
formula is:

HN^/~\^N_/"~\_NH

In the thiazins, such as methylene blue, the two benzene rings are
further joined together by a sulfur atom, forming three closed
rings of atoms. The simplest thiazin base would be:

. S _/\_NH

ACID CHROMOPHORES

1. The nitro group, ■ — NO2, as in picric acid

2. The quinoid benzene ring.

fl

which occurs in a long series of dyes, such as the indamins above
mentioned, the xanthenes and the di- and tri-phenyl methanes,
which include many well known stains, such as rosolic acid,
fuchsin, methyl green and the methyl violets. A typical triphenyl
methane formula is that of pararosanilin base:

H2N

C_/~"\_NH
H2N

General Nature of Dyes 31

LEUCO COMPOUxNDS

The different chromophores differ considerably from one another,
but they all have one property in common. In the language of
chemistry, they all have unsatisfied affinities for hydrogen; or in
other words, they are all easily reducible, for combining with
hydrogen is the opposite of oxidation and is, therefore, reduction.
The nitro group may be reduced to an amino group; in the azin
group the bond between the nitrogen atoms may break and two
hydrogen atoms be taken on; while in the various chromophores
with double bonds (such as the quinoid ring) the double bond may
break and hydrogen atoms become attached to the valencies thus
freed.

Now in every case this reduction destroys the chromophore
group, and as a result the compound loses its color. In other words
a dye retains its color only as long as its affinities for hydrogen are
not completely satisfied. These colorless compounds are known
as leuco compounds; thus fuchsin yields leuco-fuchsin on reduc-
tion, and methylene blue reduces to leuco-methylene-blue. For
example:

H.N / \ CH,

H3N

NH.

fuchsin leuco-fuchsin

Ordinarily this reaction is reversible under conditions favoring
oxidation. It is of especial significance to the bacteriologist, as
dyes can often be used as indicators of reduction.

Certain dyes form a still different type of leuco compound, often
called a "leuco-base." We have seen that the basic dyes ordinarily
occur as salts of some colorless acid; now, in the case of certain
dyes, notably the tri-phenyl methanes and xanthenes (Chapters
VII and VIII), as soon as the acid radical is removed, the com-
pound becomes colorless. This is because a rearrangement of the
atoms in the molecule takes place upon neutralization so as to
give, not the true dye base, but a compound known as a carbinol
(see p. 137) in which the chromophore does not occur. Thus the
theoretical base of fuchsin which should be obtained upon removal
of the acid radical is:

CH3

\
H2N_/— \

HoN

C^/~\^NH20H

./

32 Biological Stains

The compound actually formed, however, is the pseudo-base or
carbinol :

/~\_NH2
CH3 0H/\_/

H2N_/— \_C

\ / \

"\_NH2

y

In this compound, it will be readily seen, there is no chromophore;
hence it is colorless. These pseudo-bases are of little significance
to the biologist, but they are of importance to the dye manufac-
turer as intermediates in the preparation of dyes.

.In the case of many acid dyes the chromophore is similarly
broken by a rearrangement of the atoms which occurs on neutrali-
zation. This reaction is ordinarily very readily reversible and
makes such dyes useful indicators of acidity. It is discussed more
fully under acid fuchsin (p. 154) and phenolphthalein (p. 198).

In the case of basic fuchsin, a still different type of leuco com-
pound can be obtained, which has recently acquired considerable
importance, both in cytology and in histochemistry. When this
dye is reduced with sulfurous acid or a sulfite, a type of leuco-
fuchsin is produced which is known as Schiff's reagent, and has
been used by chemists for many years as an indicator of the pres-
ence of aldehyde. Originally this reagent was thought to be no
different from the leucofuchsin referred to on the preceding page;
but when its use as a histochemical reagent was first introduced it
was noticed that the restored color, after contact with the alde-
hyde-like constituents of cells, was somewhat violet, rather than
red. This seemed to indicate that some other chemical change in
the dye takes place in addition to the reduction. It is now be-
lieved that the sulfite radical combines with the reduced compound
in some way; and the resulting compound, Schiff's reagent, is now
generally called fuchsin-sulfurous acid, rather than leuco-fuchsin.
The importance of this reagent is being appreciated more and
more, as histochemistry increases in significance; and it will be
treated more fully elsewhere in this book.

CLASSIFICATION OF DYES

On the basis of the chromophore present the simple synthetic
dyes are classified into several groups. If each of these groups
were characterized by a single color or by a few closely related
colors, dye chemistry would be a comparatively simple proposition.
As a matter of fact a single chromophore may occur in dyes of
practically all colors of the rainbow. It is ordinarily impossible to
determine, a priori, from the chemical formula of a dye what par-
ticular color the compound may have; but there is, nevertheless, a

General Nature of Dyes 33

certain general rule which correlates chemical formula with color.
In any group of compounds, the simpler ones are converted into
the more complex by substitution of radicals for hydrogen atoms.
In the dyes the substituents are generally methyl or ethyl groups,
or sometimes phenyl groups. Now the general rule is that the
larger the number of hydrogen atoms that have been replaced by
these groups the deeper the color. The tendency is for the color
of the simplest dyes in any group of homologous compounds to be
yellow, passing through red to violets and then greens and blues, as
the homologs become higher through the introduction of success-
ively larger numbers of methyl or other substituting groups. Thus
the compound pararosanilin, which is very frequently sold as basic
fuchsin is a triphenyl methane, with an amino group attached to
each benzene ring, but without any methyl groups; thus:

H /~\^NH2

H-X_/-\_C

/ \_/ \/-\_NH2
CI \^/

Rosanilin, which is similar in composition, but contains one methyl
group attached to one of the benzene rings,

CH:

!

/

H

/-

""\_

_NH2

\

-/

H N_

=/"

\_

_c

/ '

_/

" \/-

.NH,

CI

\

_/

is a red very similar to pararosanilin but with less of a yellowish
cast. Now another methyl group may be introduced into each of
the other two benzene rings, and each one successively deepens the
shade of red, so that the highest homolog of the series, new fuchsin:

CH3

/
/~\_XHo

_C

"\ NH>

CH3

has a more bluish cast than any of the others. Thus basic fuchsins
can vary considerably in their shade according to the proportions
in which these four possible components may be mixed.

It is also possible in another way to deepen the color of pararo-
sanilin still further, namely by introducing methyl groups into the
amino radicals instead of directly on the benzene rings. Thus the
methyl violets are obtained; and the more methyl groups intro-

34 Biological Stains

duced the bluer the violet, until when all six available hydrogen
atoms are thus substituted, crystal violet, the deepest of them all,
is obtained. By using three ethyl groups instead of methyl,
Hofmann's violet or dahlia is formed, which is deeper in color than
the trimethyl compound, due to the heavier groups introduced. If
three phenyl groups (i.e., the benzene ring, CeHs-) are introduced
instead of methyl or ethyl, the color is still further deepened, the
resulting dye being spirit blue. Further, it is possible to introduce
another methyl group into crystal violet, by addition of methyl
iodide (or chloride) to one of the trivalent nitrogen atoms, whereby
its valency is increased to five, and a green dye, methyl green, is
produced :

_C

CI CH3

With these facts in mind it will be seen that the grouping of dyes
as based upon these chromophores does not classify them in rela-
tion to their color. It is a useful classification, however, because it
puts together those that have similar chemical structure. The
important biological dyes, thus classified, fall into the following
groups :

A. The nitroso dves.

e.g., naphthol green B.

B. The nitro dyes.

e.g., picric acid.

C. The azo dyes.

e.g., methyl orangey Bismarck brown F, orange 0, Congo
red, Sudan III and Sudan IV.

D. The quinone-imine group, including

1. Indamins; e.g. toluylene blue.

2. The indophenols

3. Thiazins; e.g., thionin, toluidine blue, methylene blue.

4. Oxazins; e.g., brilliant cresyl blue, Nile blue.

5. Azins, including

(a) Amino-azins; e.g., neutral red.

(b) Safranins; e.g., safranin 0, magdala red.

(c) Indulins; e.g., nigrosin.

General Nature of Dyes 35

E. The phenyl-methane dyes, including

1. Diphenyl-methanes, e.g., auramine 0.

2. Diamine triphenyl methanes; e.g., malachite green,
brilliant green, light green.

3. Triamino triphenyl methanes; e.g., basic fuchs in, acid
fuchsin, methyl violet, gentian violet, methyl green, anilin
blue.

4. Hydroxy triphenyl methanes (rosolic acids); e.g.,
aurin, red corallin.

5. Diphenyl naphthyl methanes; e.g., Victoria blue R.

F. The xanthene dyes, including

1. Pyronins; e.g., pyronin Y and B.

2. Rhodamines; e.g., rhodamine B.

3. Fluoran derivatives; e.g., eosins, erythrosin, rose bengal.

4. Phenolphthalein and the sulphonphthaleins.

5. The acridine dyes, e.g., acriflavine.

G. The anthraquinone dyes.

e.g., alizarin.

H. The thiazole dyes,
e.g., titan yelloiv.

I. The quinoline dyes,
e.g., pinacyanol.

DYE NOMENCLATURE

Very little system has been used in naming dyes, and as a result
their nomenclature is extremely confused. Generally the manu-
facturer of a dye which he thinks is new or which he wishes the
public to consider a new dye sells it under a new name which is not
intended to give any clue as to the nature of the dye. If the
manufacturer knows that the name is a mere synonym of one al-
ready in use he does not say so, for he wishes to encourage the sale
of his own product rather than that of some other dye maker.
Accordingly it has been left for others, who are not financially
interested, to work out the synonymy of the dyes; and the list of
names that are found to apply to a single dye is sometimes amaz-
ing.

With the dyes in general so unsystematically named, it is natural
that the same confusion should reign in the nomenclature of bi-
ological stains. This confusion is very unfortunate, for it often
misleads the biologist as to just what he is doing. For example,
some histologist may have on hand a bottle of stain labeled dahlia
and he may find it useful for some new technic, which he publishes;
while another may propose for an entirely different technic the

36 Biological Stains

stain Hofmann's violet. Then a third laboratory worker may redd
both articles and wish to try both methods; so he accordingly
orders both dahlia and Hofmann's violet. His dealer, who may
be unacquainted with dyes, will very likely send him a bottle bear-
ing each name, and the purchaser has no easy way of discovering
that the two are identical; so he may continue for years to use the
two stains for different purposes, misled by their labels and think-
ing them distinct. The manufacturers and dealers in stains have
sometimes encouraged this confusion by their practice of taking
care to have the label on the bottle agree with the name used in
the customer's order, regardless as to what the usual name for the
dye may be.

An attempt to relieve this confusion has been made by the Com-
mission on Standardization of Biological Stains (1923f) by publish-
ing a list of biological stains with their best known synonyms. In
each case one of the names is listed as a preferred designation.
Sometimes general usage made it easy to select one name as the
preferred one; but in other instances the selection was more or less
arbitrary. This same list, with a few revisions in the way of ad-
ditions and corrections, is given in the appendix of this book (p.
252; see p. 106 of first edition). A few new stains have been
added to this list, but essentially it is the same as published in
1923. The chief changes have been in the list of synonyms, which
has been* revised to omit names that are obsolete and have no
present meaning. With but one or two exceptions the preferred
designations are still the same as in the first list.

DYE INDEXES

There have been two important indexes of dyes, one published
in Germany (Schultz' Farbstofftabellen) the other in England (the
Colour Index of the Society of Dyers and Colourists). Seven edi-
tions of the former appeared up to the time of the second World
War, while only one edition (1923) has yet appeared of the latter.
It was natural that the earliest efforts to index dyes should have
been made in Germany as there was almost no dye industry out-
side that country until 1914; but the 1923 edition of Schultz was
so much less complete than the Colour Index which came out the
same year that there has been a recent tendency to follow the latter
rather than the former. In the second edition of the present book,
therefore, the Colour Index numbers of the various dyes were listed
instead of the Schultz numbers.

The seventh Schultz index was as complete as the Colour Index,
with an entirely new system of index numbers assigned to the dyes,
agreeing neither with that in the other publication nor with earlier
editions of Schultz. Considering how often it is desirable to refer
to a dye by an index number, it is unfortunate that these numbers
cannot be standardized in some way by international agreement.

General Nature of Dyes 37

In the present edition of this book the Colour Index numbers
(abbreviated C. I. No.) are still used, so as to avoid confusion and
to make the present index numbers agree with the list previously
published. The Schultz number of each of them is given for
reference purposes, however, in the lists in the appendix, pp. 252,
260.

Although there has been as yet only one edition of the Colour
Index, a new one is now in course of preparation by the Society of
Dyers and Colourists in England, with the cooperation of the
American Association of Textile Chemists and Colorists. The
new edition is to be entirely different from the first, consisting of
more than one volume; and it will contain many more dyes than
ever before listed. Although work on it has been in progress for
some time, no probable publication date has yet been announced,
as the task is proving a large one. The compilers of this Index
are fully aware of the confusion caused by frequent changes in
index numbers; so it is their plan to adopt an entirely new system
in the second edition, with numbers so different in appearance from
the old ones that they cannot be confused with them. The idea is
to have the new numbering system one that will allow for expan-
sion anywhere and thus avoid the necessity of continual change
with each new edition. If this system works out as hoped, it is
possible that in the next edition of this book, the new C.I. numbers
can be followed.

At the time of the fourth edition of this book, the plans for the
revision of the Colour Index were not known, and it was feared that
as soon as a second edition of that Index appeared, further refer-
ence to the old C. I. Numbers would be distinctly confusing. In
anticipation of this situation the 4th edition of the present book
included at the head of the description of the various dyes, a
series of numbers designed particularly to apply to biological
stains. This policy is continued in the present edition. The new
numbers are printed in a smaller type than the Colour Index num-
bers and no special significance is being given to them. It is
planned, however, to use them in the future under the designation
"Biological Stain No." only if a new edition of the Colour Index
makes it necessary to abandon the "C.I. Nos." at present in use
without furnishing more satisfactory index numbers in their place.
It is hoped that because of the relatively small number of dyes used
as biological stains and the fact that the "B.S. Nos." at present
selected are not entirely consecutive, there would be no need of
changing the significance of each number whenever a new edition
appears. Whether or not to use them cannot be decided until the
new Colour Index appears.

DYE SOLUBILITIES
Textile dyes are never of a high degree of purity. Some of the
impurities are accidental; others are added intentionally so that

38 Biological Stains

dyers can obtain the desired shade without having to measure out
dyes in very small quantities. Inasmuch as the early biological
stains were textile dyes without much, if any, modification, it is
natural that some of them should also have been of low dye con-
tent, and also that different batches should have been of various
degrees of purity. In general the present dyes are much more
pure than those available before the war of 1914-18. This makes
it difficult to prepare stain solutions identical in strength with those
prepared before that time.

There are two general types of stain formulae: in one a definite
weight of dry dye is specified; in the other a certain volume of a
saturated (generally alcoholic) solution of the dye. Each type of
formula has its own possibilities of error; and to appreciate the
problem it is necessary to understand certain facts in regard to the
solubilities of dyes.

The error inherent in the first type of formula is plain at a
glance. If two different staining solutions are made up containing
1 g. per 100 ml. of dry methylene blue, and in one case the actual
dye content of the dry stain is 90 percent, while in the other only
55 percent (a difference actually observed in samples at one time
on the market), it is plain that the two solutions must differ greatly
in their strength. For this reason an early recommendation of the
(commission (1928b) was that formulae of the second type be pre-
ferred, on the assumption that a saturated solution of a dye would
be more likely to be of constant dve content than different lots of
dry stain bought in the market.

This recommendation, however, was made without complete
understanding of the actual facts of the case. The amount of a
dye that will go into solution in either water or alcohol depends
upon the amount of mineral salts present. If a dye contains a
large percentage of sodium chloride, for instance, a saturated solu-
tion will be of considerably lower actual dye content than if the
dye were free or nearly free from salt; the sodium chloride prevents
the solvent from taking up as much of the dye as it would nor-
mally. For this reason two staining solutions each containing 10
percent by volume of a saturated solution of the two methylene
blues above mentioned would be quite different from each other
in actual dye content, although possibly more nearly alike than if
they had been prepared with identical weights of the dry stain.

As soon as the facts were fully understood the Commission
(1933e) modified its recommendation. It is plain that the only
way two stain solutions can be made identical, if different batches
of stain are used, is to make them up on the basis of the weight of
actual dye present in the stain samples employed. For some time
now, all staining formulae recommended by the Commission have
been put in such a form as to call for a definite weight of dye of a
specified dye content — thus enabling the user to recognize any

General Nature of Dyes 39

correction he has to make if he has a dye sample of different dye
content. Many individual writers, however, especially writers of
text books, have not followed this system. There are various
reasons such a plan has not been generally adopted. The chief
reason is that there is much variation in the dye content of in-
dividual batches, and it is obvious that the staining formulae can-
not be generally put in the form recommended by the Commission
unless the manufacturers print the actual dye content of the batch
of stain on the container in which it is sold. Until about 1930 this
was never done. Since about that time, however, the Stain Com-
mission has been issuing its certification only when the dye con-
tent is printed on the label, except in instances where the neces-
sary analytical methods are not available. This policy has grad-
ually been changing the situation. At the present time almost all
stains on the American market, except natural dyes, compound
dyes like the blood stains, and a few complex dyes for which good
analytical methods are still unavailable, are labeled as to their dye
content. In all Commission publications (including Stain Tech-
nology) all formulae are given in terms of grams of Commission
certified stains (unless the dye in question has never been certi-
fied), and as certification is now issued only on samples that vary
within narrow limits, this amounts to recommending definite
weights of actual dye. Gradually authors of papers published
elsewhere are getting into the same habit; so the situation is much
more satisfactory than a few decades ago.

In the present edition of this book there are listed, both under
the individual dyes and in Table 6 in the Appendix, the solubilities
of pure dyes in 95% alcohol and in water at 20° of the most com-
monly used stains. These data were originally published by
Holmes (1927, 1928, 1929) and are based on determinations made
by a cooperating laboratory of the U. S. Department of Agricul-
ture. The author of these papers experienced considerable diffi-
culty in obtaining the information given therein because of the
strong effect of impurities upon the amount of dye capable of going
into solution. The commercial samples of the dyes are rarely pure,
and Holmes found it necessary to recrystallize each sample before
determining solubility. This fact, therefore, must be taken into
account in interpreting the figures. To obtain a solution of many
dyes as strong as the figure given in the table, the dye sample em-
ployed must be strictly pure. As this is practically never true,
the user of stains can rarely expect to make a saturated solution of
the theoretically possible strength. These data nevertheless are
useful in indicating the amount of stain to employ in order to
obtain a saturated solution.

The data given in Table 7 were compiled in the hope of furnish-
ing information of more practical value to users of stains than those
published by Holmes; these were obtained through the courtesy

40 Biological Stains

of the American Pharmaceutical Association. In this table there
are listed the solubilities of 50 samples representing 25 com-
mercial certified stains. They are of interest In showing approxi-
mately how much deviation to expect between commercial samples
of the same dye and about how nearly the solubilities of such
samples approach those of the recrystallized dyes listed in Table 6.
Frequently the data In Table 7 may prove of more assistance In
determining how much dye to employ in making a saturated solu-
tion than those given In Table 6.

It would, of course, be still more useful if the solubilities in
water and in alcohol of each batch could be printed on the label.
Unfortunately, however, no practical method of furnishing this
information to the manufacturers and of publishing it on the labels
has yet been devised.

INFLUENCE OF IMPURITIES ON INTENSITY OF STAINING

Impurities present in any dye sample or in the solvent not only
Influence the solubility of the dye but may have a great effect on
the intensity of staining. An impurity present may alter the
H-ion concentration of the staining fluid; while acid dyes stain
better In more acid solutions, and basic dyes in more alkaline solu-
tions. If, moreover, the impurity is a mineral salt or the salt of an
organic acid, it may have an effect on intensity of staining even
if the reaction of the solution is not altered. Theories to explain
these facts are discussed in Chapter III.

The practical lesson from this is that poor staining results with
any dye sample are as often to be explained by the presence (or
absence) of impurities In the dye, or in the water used as a solvent,
as by some irregularity in the dye itself. In many instances the
impurities normally present In some dye may be necessary to
assure its proper behavior as a stain; and too great effort in the
way of purification may be detrimental. This seems to have been
unquestionably the case in respect to rose bengal. It has been
found (Conn and Holmes, 1928) that a sample of the latter dye,
too highly purified for good results if dissolved in distilled water,
may stain satisfactorily in tap water containing a certain amount
of calcium. It may well happen that the user of some dye which
gives too weak staining can Increase its Intensity of action by add-
ing minute amounts of some mineral salt to the staining solution
or by altering the H-ion concentration. In trying this experiment
great caution must be employed, for large effects are sometimes
produced by minute changes, and it is easy to bring about too
weak staining on the one hand, or overstaining on the other if too
much of any salt, acid, or base be added.

General Nature of Dyes 41

ASSAY METHODS

When the project of stain standardization was undertaken, in
the early '20's, it was realized that reliable methods of assay were
needed. They were so definitely lacking at that time that in the
early certification work, primary stress was laid on performance of
the batches tested; chemical and optical assay methods were at the
start more or less incidental. This was regarded as an unsatis-
factory and temporary expedient; but it was adopted because
tests for performance were then regarded as more reliable than the
assay methods then available.

These methods were crude. Dye content had to be determined
bv means of the rather trickv method of reduction with titanous
chloride. This procedure depends on the fact that reducable rad-
icals are present in almost all dj^es, so that the dye strength of a
sample can be determined by TiCls titration, provided that: (1)
the end point is sharp; (2) the molecular weight of the dye is
known. These provisos make the method sometimes inapplicable.
It often happens that the correlation is very poor between titra-
tion and colorimetric comparison of various samples of the same
dye. As a result considerable research is often needed to establish
a reliable assay method; nitrogen determinations or other more
absolute procedures must be resorted to in order to establish a
satisfactory norm for actual dye content determinations.

In addition to assay for dye content, the spectrophotometer
was adopted quite early in the work for determining whether or
not any sample was true to type. The original spectrophotom-
eters (see Chapter III) were visual instruments, and it was quite
laborious to make accurate readings. The later introduction of
photoelectric spectrophotometers has made for greater accuracy,
but it has also necessitated some research before standards based
on the new type of instrument were well established.

One advantage of the photoelectric instruments is the greater
ease in obtaining with them a reading of the density at the peak of
adsorption (Dp). This has made it possible to use the spectro-
photometer not only for determining the nature of the dye but
for comparing the strength of various samples in cases where the
titration method has proved inadequate.

The methods at present adopted in the Commission assay labo-
ratory are given in the appendix of this book (pp. 292-328). Con-
stant efforts are being made to improve the methods, and it is
realized that they are much better than those adopted in the '20's.
Nevertheless they are still unsatisfactory as methods for evalu-
ating the performance of stains; and it is still necessary to con-
tinue the practice of testing them in actual use.

HISTOCHEMICAL REAGENTS

The possibility that dj^es might some day serve as histochemical
reagents was realized quite early. The well known fact that basic

42 Biological Stains

dyes have greater affinity for nuclear substance, acid dyes for
cytoplasm is enough to suggest this possibility to anyone; but for
a long time the matter remained in the realms of theory, without
much concrete fact on which methods could be based.

Undoubtedly the first real stimulus to histochemical technic
came from Feulgen and Grossenbeck's application of the Schiff's
reagent for aldehyde to cytological structures. (See p. 150).
Feulgen was not unaware of what he had accomplished, as shown
by the fact that he named the granules thus stained "nucleal" the
terminal syllable indicating their supposedly aldehyde-like nature.
That was about as far as one could go in those days; not until the
various nucleic acids came to be recognized and localized in the
cell could the specificity of the Feulgen reaction or the nature of
the bodies stained by it be learned.

It was probably ten years or more after Feulgen's work before
the reaction named after him became well enough established so
that the Stain Commission regarded as a sufficiently important
use for basic fuchsin to serve as one of the criteria of certification.
It was realized that not all basic fuchsins were suitable for use in
the technic; and for some time it seemed an unnecessary refine-
ment to insist that a certified fuchsin should meet this requirement
when the dye was so seldom used for the purpose. Now, however,
the situation has changed greatly, thanks to the rapid development
of histochemical technic, and no basic fuchsin unsatisfactorv in the
Feulgen procedure would be acceptable.

At present the efforts of histochemists are being directed toward
finding other reagents that are specific for the same or other cyto-
chemical features. Some of the reagents are dyes, others are not
dyes but develop color in the presence of the chemicals with which
they react. The proposal has been made to extend the principle
of stain certification to cover some of these reagents. No definite
plans to that effect have yet been made, but the project is being
given careful consideration at the present time.

CHAPTER HI
THE THEORY OF STAINING

THROUGHOUT the other chapters of this book an effort has
been made so far as possible to avoid theoretical discussions.
Although they contain some statements the truth of which
cannot be regarded as fully established, as in the case of the chemi-
cal composition of some of the dyes, the discussion in general is
confined to observations and to chemical information for which
there is good authority, without any attempt to introduce ex-
planations of a theoretical nature. The present chapter, dealing
whollv with theorv, is included because it is felt that a brief state-
ment of some of the most probable theories to explain stainmg
may be of value in assisting the histologist in the intelligent use of
stains for his purposes.

A long theoretical discussion of this subject might be included
here, basing it upon the lengthy arguments supporting the various
theories that have appeared in the literature. Such a detailed dis-
cussion, however, would probably be of little value. Hence this
chapter is confined to a bare outline of the important points of the
different theories.

Theoretically the dyeing of textile fabrics and the staining of
microscopic structures are the same. In one case only the gross
effects are observed, in the other the microscopic details. Any
theory, therefore, that will explain the details of microscopic stain-
ing will be fully adequate to account for dyeing in bulk.

The majority of such theories have depended upon either physi-
cal or chemical phenomena rather than on both simultaneously.
It would seem at first thought that the dyes combine so firmly with
the tissues stained by them that the phenomenon must be a
chemical one; but the exponents of physical theories have taken
pains to show that all the observed facts can be explained on a
physical basis, and that some observations are hard to explain if a
chemical union between tissue and dye actually takes place. In
a chemical union a new substance is formed which does not neces-
sarily have the properties of either substance entering into its
formation, and it is ordinarily impossible to recover the original
substances by means of simple solvents. When tissue is stained
there is no evidence of any new substance having been formed, the
colored tissue merely taking on one of the characteristics of
the dye (color) in addition to the properties which it originally
possessed; it is, moreover, ordinarily possible to extract all or near-
ly all of the color by sufficiently long immersion in water, or by the
fairly brief action of alcohol. Another observation which points
against chemical action is that the tissue never removes the dye
completely from solution, even though very dilute; whereas or-

48

44 Biological Stains

dinary chemical reactions tend to continue until one of the com-
ponents of the reaction is exhausted. Although these facts are
not incompatable with chemical explanations, the original expon-
ents of the physical theories regarded them as full refutation of the
possibility of chemical action.

The original dispute concerning, physical and chemical theories
of stain action is now rather obsolete. Much of it was based on
a priori argument or on insufficiently understood evidence. Since
1945 there have been various investigations of a more scientific
nature, due largely to the growing importance of histochemistry,
which are continually bringing out new facts concerning the
mechanism of staining. The most recent pubUcations on the sub-
ject do not try to differentiate between physical and chemical
theories.

The Physical Theory. It was formerly pointed out that all or-
dinary dyeing or staining phenomena can be explained on a physi-
cal basis. There are three factors, all purely physical in nature,
which together or separately may explain many, if not all, stain-
ing phenomena. In the first place, as nearly all substances stained
are more or less porous, simple physical forces such as capillarity
and osmosis can account for penetration of the dye into the interi-
or of the tissue. In the second place, the action of acZsorption
can account for many staining phenomena, even for much of the
selective staining with which the biologist is familiar. In the
third place, a dye may penetrate some cellular element by absorp-
tion, remaining there in a state of solid solution. Any one or
all of these three forces may operate in any given instance; and
even the exponents of the physical theory of staining differ in
the amount of weight given to each of them. The penetration
of the dyes into the tissue by osmotic action is generally ad-
mitted; but some students of the subject favor adsorption, others
absorption, as the primary explanation of staining phenomena.
For a concise discussion of the physical explanations, especially
the solution theory, see Holmes (1929 b).

The absorption or solution theory is very simple and the action
of some dyes on some kinds of cellular tissue in the presence of
mineral salts suggests that this factor is important, at least in some
instances. It is also supported by the fact that a dye causes the
tissue to become the same color as the dye shows in solution, but
not necessarily the same as it shows in its dry form. Dry fuchsin,
for example, is green; its solution, however, is red, and so are
tissues stained by it, no matter how completely they may be dried.
It is nevertheless difficult to explain all staining phenomena, par-
ticularly differential staining, on the basis of solution. There are
admittedly some special staining phenomena, like fat-staining,
where simple solution is the obvious explanation; these, however,
form a special case.

Theory of Staining 45

Adsorption, on the other hand, furnishes a very satisfactory
explanation even of much differential staining, and many once
claimed that all staining phenomena might be accounted for in this
way. Adsorption is the property possessed by a solid body of
attracting to itself minute particles of matter from a surrounding
fluid; these particles may be compounds suspended in the fluid,
or they may be ions incapable of existing independently except in
solution. The principle of selective adsorption is well known to
physical chemists, whereby certain ions may be adsorbed by
certain substances much more readily than by others. Equally
well known is the fact that the rate of adsorption of any ion is
strongly influenced by the presence of other ions in the solution,
and that an especially profound influence is exerted by the re-
action of the solution, in other words by the concentration of free
hydrogen or hydroxyl ions in the fluid; the influence of the latter
upon the adsorption of basic ions being exactly the opposite from
its effect upon the adsorption of acid ions. Such principles as
these, well established in physical chemistry, may offer an explan-
ation of such phenomena as the differential staining of different
cellular elements, action of mordants, variations in rate of staining
with changes in the salt content of the staining solution, and in-
fluence of H-ion concentration upon the color assumed by tissue
when exposed to the action of both acid and basic dves. Bavliss
(1906) developed this particular phase of the physical theory into
what he calls an "electrical theory of staining." See also Parks
and Bartlett (1935a and b).

Some of those who held in general to the physical theory of
staining admitted that these simple phj^sical phenomena alone can-
not explain everything, as for example, instances in which a dye
penetrates different cell elements equally readily, but can be easily
extracted from some of them while scarcely at all from others. It
was assumed, therefore, that the dyes penetrated the cells by mere
absorption and diffusion, but were in some cases precipitated there
by acids or bases, or other chemical reagents present, thus pre-
venting their extraction by simple solvents. Such a theory admits
the possibility of chemical action without assuming an actual chem-
ical union between the dye and the tissue.

This precipitation theory would furnish a satisfactory ex-
planation of the action of mordants. It is well known that certain
tissues, that stain feebly if at all with certain dyes, "take" these
same dyes deeply if previously treated with the proper chemical.
Such a mordant, already present in the tissue, might well cause
the precipitation of the dye inside the cell walls. On the other
hand the action of mordants may be equally readily explained if it
is assumed that staining takes place by means of adsorption or as
a process of solution. In the first place, all mordants contain
ions that are known to have a decided influence upon the rate of ad-

46 Biological Stains

sorption, and their action may be thus accounted for; they also
are nearly always substances that decrease the solubility of any
dye and thus increase the readiness with which a dye may enter
into solution within the tissue to be stained, if one accepts the
solution theory. In other words, the action of mordants can be
accounted for by any of the theories of staining.

It is, moreover, frequently pointed out that there is no sharp
distinction between chemistry and physics, and in such delicate
reactions as those involved in staining, w^e may be well in the
borderland between the two branches of science, where it is im-
possible to say that a given phenomenon is purely physical or
purely chemical. There are, however, certain chemical principles
distinctly different from the physical ones just mentioned, that
may well enter into the phenomenon of staining; and it is these
that were considered most important by the exponents of the chem-
ical theory.

The Chemical Theory. It was claimed on behalf of the chemical
theory that just because physical forces alone can explain the
facts one is not justified in assuming that chemical unions do not
take place when the opportunity for them is present. It is agreed
that some parts of the cell are acid in reaction, others alkaline;
and it is a well-known chemical principle that the former w^ould
tend to combine with the cations in solutions with which they
come in contact, the latter with the anions. Now inasmuch as in
certain dyes the color exists in the cation (basic dyes) and in
others in the anion (acid dyes), it is natural to expect chemical
combinations to take place between dye and tissue, depending
upon the reaction of the latter. Arguments for the physical
theory which exclude chemical action must furnish strong proof
that no chemical union occurs; and those who favored the chemical
theory claimed that such proof is Licking. That the stained tissue
does not present any characteristics to the eye not possessed by
either tissue or dye before staining does not prove that no new
substance has been formed, nor is this claim refuted by the fact
that suflSciently long action of solvents removes the color. Alcohol
and even water are not absolutely inert chemically and may with-
draw the dye by chemical instead of physical action; the very
length of time necessary to remove the color completely (some-
times so long as to allow bacterial decomposition of the tissue)
indicates that a rather strong union between dye and tissue has
taken place. As to the fact that a tissue which has a strong affinity
for some particular dye never withdraws that dye completely from
a very dilute solution, those who favored the chemical theory pointed
out instances where chemical reactions are known to take place
and yet to stop before either component is exhausted. The failure
to exhaust the dye completely in a surrounding solution may
merely mean that an equilibrium has been reached; chemical

Theory of Staining 47

action, moreover, is strongly indicated by the fact that in dilute
solutions the tissues take up relatively larger quantities of the dye
than in concentrated solution. Accordingly the observation that
not all the dye is withdrawn from the solution is a poor argu-
ment against chemical action.

Furthermore, if adsorption is assumed to take place only at a
specific site (i.e. at the COOH 017 NH3 group), the physical and
chemical concepts merge into one and cannot be distinguished
from each other. It has been shown, for instance, that cells in
which the COOH groups are tied up in methyl esters do not stain
well with basic dyes. This observation is hard to explain on a
purely physical basis; to explain it bj- adsorption requires the as-
sumption that the phenomenon occurs only at a specific chemical
site. Such an explanation comes quite close to assuming chemical
action.

In brief, the chemical theory of staining is that the tissues have
certain definite chemical affinities which are satisfied by the chem-
ical affinities of the dyes; therefore, when the tissue is put in a
solution of the dye the latter combines with those portions of the
tissue or of the individual cells which have the proper chemical
nature. This theory, it will be seen, is especially well adapted to
explain the differential staining which takes place when we find
a certain stain acting only on the nuclei. It must be remembered,
however, that it is merely a theory. As a matter of fact, the
probabilities are that staining is both a chemical and a physical
phenomenon.

The chemical theory of staining depended largely upon the
question of the acid or the basic character of the dye molecule. It
will be recalled that all ordinary dyes are encountered either as
sodium or potassium salts of dye acids or as dye salts of colorless
acids, the former being the acid dyes and the latter the basic dyes;
while certain compound stains are neither acid nor basic dyes, in-
asmuch as the property of color exists in both the anion and the
kation.

The fundamental principle involved in this theory is that
certain parts of animal or plant cells are acid in character and
hence have an affinity for the basic dyes. The nuclei of the cells,
or especially the chromatin within the nuclei, are assumed to be
acid in character (due largely to their constituent nucleic acid),
and there is no question but that they have a strong affinity for
basic dyes; while the cytoplasm has an affinity for acid dyes and is
assumed to be basic in character. (See Stearn and Steam, 1929-
30.)

This theory assumes that the acids and bases which go to make
up body tissue are ordinarily amphoteric, capable of acting as
bases in acid solutions and as acids in basic solutions, the H-ion
concentration at which any such compound changes from an acid

48 Biological Stains

to a base in its action being known as its isoelectric point. It also
assumes that these compounds, although insoluble, act as though
they were electrolytes dissolved in any fluid in which they are
immersed. Hence, on this assumption, such a compound acts as a
base or as an acid in any staining solution according to whether
its isoelectric point is below or above the H-ion concentration of
that solution. The chemical theory therefore postulates that any
cellular element takes a basic dye if the H-ion concentration of the
staining solution is below its isoelectric point, an acid dye if it is
above that point.

Now, exactly such a phenomenon as this is to be observed in
staining any tissue. With very acid staining solutions even the
nuclei take the acid dyes; if one employs successively a series of
solutions of decreasing H-ion concentration, the aflinity of the
nuclei for the acid dyes rapidly becomes less ; till at a fairly definite
reaction, usually in the region of pH 4, they lose their affinity for
acid dyes and take the basic dyes. In solutions near neutrality,
therefore, the nuclei take basic dyes, the cytoplasm the acid dyes.
Finally at a point considerably to the alkaline side of neutrality
even the cytoplasm takes the basic dyes. Such an observation is
interpreted to mean that the isoelectric point of the nuclei lies
considerably to the acid side of neutrality, that of the cytoplasm
considerably to the alkaline side. (This is merely another way
of making the statement, given two paragraphs above, that the
nuclei are acid in character, the cytoplasm basic.) Now assuming
that this interpretation is correct, it might be possible to determine
the isoelectric points of different parts of the cell by staining at
different reactions. Stearn and Stearn (1928), who were among
the leading exponents of the chemical theory in the days of the
most heated arguments on the subject, tried to determine in this
way the isoelectric points of different species of bacteria. They
concluded it to be the pH-value of the crossing point of the staining
curves of acid and basic dyes. This idea has been distinctly dis-
credited of recent years. For one thing, staining determinations
of any protein must be made on fixed tissue; and the isoelectric
point of a fixed protein may well be very different from that of the
fresh material. Also, as Singer (1952) points out in his review of
the factors which control staining, the position of the crossing
point of the staining curves is determined by various other factors,
and does not definitely indicate the isoelectric point. He con-
cludes, nevertheless, that although this point cannot be precisely
defined by pH staining characteristics, it is possible to compare
the curves of different proteins and to learn therefrom the relative
position of their isoelectric points. Such results show that there
is a definite tendency for cationic dyes to unite with anionic ele-
ments of the tissue, and vice versa.

This is not by any means the whole of the chemical theory of

Theory of Staining 49

staining. It is difficult to explain wholly on the basis of various
isoelectric points the fact that certain basic dyes have stronger
affinities for certain parts of the nuclei than for others, and that of
the various cytoplasmic structures outside the nucleus some are
more readily stained by certain acid dyes and some by others.
In the Flemming triple stain for example, which employs the
acid dye orange G and the two basic dyes safranin and gentian
violet, with intervening alcoholic differentiation, it is possible to
stain the chromatin with gentian violet and the rest of the nucleus
with safranin. Before such a phenomenon as this can be explained
on a purely chemical basis, it is obvious that we must have a much
more detailed knowledge of histo- and cytochemistry than is yet
available.

Discussion of the Chemical Theory. The weaknesses of the
chemical theory show up particularly in reactions such as this
triple stain. Besides the difficulty of accounting for the stronger
affinity of certain portions of the nucleus for certain basic dyes and
other portions of the nucleus for other basic dyes, there is the
difficulty of explaining the action of solvents. The differential
staining secured in the Flemming stain, and in fact in the ma-
jority of other similar procedures, is brought about not by the
staining itself but by the action of solvents which extract some
of the dyes more readily from certain portions of the cell than
from others. In case a chemical union has taken place between
the tissue and the dye, the alcohol or other solvent used must
have the effect of breaking down the chemical compound formed
between the tissue proteins and the dye molecule, or else it must
actually dissolve out the compound in question. It is, however,
difficult on the one hand to conceive of alcohol breaking down
such a compound; while, on the other hand, if the compound
formed is dissolved out of the tissue, it is hard to understand how
restaining of decolorized structures is possible.

A further difficulty of the chemical theory arises from the fact
that it assumes ionization of the compounds stained, while the
tissue is not in solution. Thus, the hypothesis above given as-
sumes a definite isoelectric point for the protein of the tissue, and a
physical chemist defines this point as the reaction where the min-
imum dissociation occurs. Now a protein occurring in a solid form
as in sections of fixed tissue can hardly be expected to dissociate,
and accordingly, it is a question whether it can properly be said to
have an isoelectric point. Under the chemical hypothesis, in short,
it is very difficult to conceive how an insoluble solid can behave
as an electrolyte and take part in the reactions assumed for it.

One can, however, assume that differential staining indicates
differences in chemical nature of the different parts of the cell with-
out adopting the generally accepted chemical theory. Even if
the dye is actually taken up by a process of adsorption, this pro-

50 Biological Stains

cess will be greatly influenced by the chemical nature of the differ-
ent parts of the cell. Substances of an acid character would ad-
sorb basic dyes more readily, and retain them in the adsorbed
state more firmly than they would acid dyes, and substances of a
basic character would adsorb acid more readily than basic dyes.
Amphoteric substances are usually acid or basic in their prevailing
character. Acid substances such as cell nuclei exert a decided
chemical affinity for basic dyes and accordingly adsorb them
readily. They have, however, inferior degrees of affinity for acid
dyes and in order to obtain staining with such dyes it is only
necessary to convert them into a favorable physical form for ad-
sorption. Basic substances, on the other hand, such as cytoplasm,
although ordinarily stained only by acid dyes, may be stained by
basic dyes if the latter are employed in a favorable physical form.
Favorable conditions for these unusual types of staining are ob-
tained merely by altering the reaction of the staining solutions.
Thus one does not have to assume chemical combination between
dye and tissue in order to account for the different type of staining
obtained in solutions of varying pH value.

It is extremely easy to theorize in regard to the mechanism of
staining but very difficult to propose any conclusive argument.
Dye chemists have obtained actual quantitative data as to with-
drawal of dyes from solution by the fibers of which textiles are
made up, which point very strongly to the process being physical
rather than chemical. These conclusions are not directly appli-
cable to biological staining for two reasons : In the first place fixed
tissues are decidedly different from the animal or vegetable fibers
studied by the dye chemists and quantitative data seem to be
lacking as to how such tissue removes any dye from solution.
In the second place, staining effects are in most instances brought
about by the action of decolorizing agents after the staining has
taken place, and this introduces a factor which dye chemists have
not had to take into account in explaining the affinity of textiles
for dyes.

Evidence is still lacking, in fact, to prove or to disprove any
of the physical theories on the one hand or the chemical theory
on the other. The difference, perhaps, is not one of immense
importance. All of the theories teach about the same practical
lessons as to the type of dye to select for any particular purpose
and as to the influence of salt content or H-ion concentration of
staining solution or tissues upon the rate or intensity of staining.
The user of biological stains should know enough about the vari-
ous theories of their action to understand the influence of ionic
concentration upon basic dyes on the one hand and acid dyes on
the other; but realizing that the same phenomena can be explained
by more than one theory, he can safely leave to the physical chem-
ists all discussion of the mechanism involved.

Theory of Staining 51

When the first edition of this book appeared, in 1925, frequent
papers on the theory of staining were being written by biologists.
Today, although much valuable work on the mechanism of stain-
ing is being done, it is ordinarily recognized that the findings
relate only to specific cases, and arguments about the general
theory of staining, as then understood, have almost disappeared
from the literature. This is partly perhaps because as we know
more we theorize less ; but other factors seem to be more important
in contributing to the lack of interest. Chief among them is the
realization of the great complexity of staining processes, and of the
necessity of both chemical and physical theories to explain them —
a complexity so great that, until we possess many more facts than
at present, our theories are little better than guesses.

A good illustration of the modern way of treating the subject is
the recent review paper by Singer (1952). In this paper the author
mentions the various theories without showing special favor to any
one of them, but gives his attention to the various controllable
factors which influence staining of tissues. He points out that the
extensive literature of the early part of this century on the theory
of staining has brought out many facts of value relating to the
interaction of dyes and tissue proteins, and that this interaction
deserves further study, without theorizing as to whether it is
chemical or physical. His paper is an excellent review of the in-
formation on this subject now available.

Histochemistry. It is interesting that now, when a purely chem-
ical theory of staining is not generally accepted, specific instances
of what seem to be definite chemical reactions between dyes (or
related compounds) and tissue elements are becoming more numer-
ous. The new science of histochemistry depends on the fact that
some dyes, or uncolored chemicals which become colored under
certain definite conditions, can be used as reagents for giving in-
formation as to the chemistry of cell constituents. Although the
chemical nature of the observed reactions seems quite evident,
such findings have little bearing on the general theory of staining.
Ordinary staining follows a comparatively leisurely process of
fixation, during which degenerative changes of the tissue can take
place; while the histochemical procedure generally involves quick
freezing or instantaneous introduction into fixing reagents, or
both, to avoid any appreciable change in the nature of the constit-
uents of the tissue. It is clear that the histochemical reagents,
therefore, are given quite a different substance to act on from that
furnished the ordinary histological stains. Furthermore, the
histochemical procedures all employ specific reactions which have
little connection with the usual staining methods. An authorita-
tive text on the subject is the book by Gomori (1952).

It is true that, as long ago as the mid-twenties, the chemical
theory of staining was leading specialists in the field to speculate

52 Biological Stains

as to the possibility of dyes becoming histochemical reagents (see
Conn, 1925), but nothing like the present development of histo-
chemistry was then forseen. Histochemistry, although a fascina-
ting field from the theoretical standpoint, is neither the outgrowth
of the general chemical theory of staining, nor does it prove the
truth of the latter as a sole explanation of staining.

CHAPTER IV

THE SPECTROPHOTOMETRIC AND
CHROMATOGRAPHIC ANALYSIS OF DYES

CHEMICAL methods alone are inadequate in the analysis of
many dyes. Not only is the detailed chemistry of some dyes
obscure, but the reactions are often complicated by adulterat-
ing dyestuffs in such a manner as to preclude entire reliance on
rigorous chemical methods. Often a slight change in the arrange-
ment of atoms within the molecule will make a marked change in
the nature of the dye, while such a minor change in structure is not
always readily detected by chemical means alone. Hence the ad-
visability of employing certain physico-chemical characteristics as
displayed by the spectrophotometer in the study of dyes. So im-
portant is this study that a discussion of the principles involved is
necessary here.

The absorptive spectra obtained by the spectrophotometer are
usually characteristic of any particular dye. Quantitative as well
as qualitative data may be obtained by the spectrophotometer;
and from the combined results nearly all dyes, even though differ-
ing from each other only in very minor particulars of chemical
structure may be easily differentiated. This method is not only
rapid but is also convenient.

The method depends upon the fact that any colored substance
absor})s light of certain definite wave lengths and transmits or re-
flects the rest. The absorption spectrum is essentially the inverse
of that which is reflected or transmitted. Therefore the color of
light which reaches the eye after transmission through or reflection
from a colored substance is complementary to the color of light
absorbed by that substance. A violet dye, for example, appears
that color because of its predominant absorption of greenish yel-
low light. The absorption maximum is quite characteristic of any
dye; any two dyes having the same absorption curve (a somewhat
rare occurrence) are of essentially the same color.

The wave length is ordinarily measured in millimicrons (m/^),
or Angstrom units (A) which are O.lm^t. The visible spectrum
})egins at around 400 m/x in the violet and runs to about 750m/x in
the red. The wave lengths of the six most important colors, and
their complementary colors are given in Table 1.

A variety of terms and units have come into common usage in
connection with the practice of spectrophotometry and numerous
symbols to represent them are employed. Since there is some con-
fusion concerning the symbols used, the relationships between the
various symbols and units and definitions of the terms most fre-
quently encountered in the literature are given in Table 2.

The color of substances is ordinarily of complex origin, depending

54

Biological Stains

upon the absorption of light in varying degrees, over an extensive
spectral range. Whereas the unaided eye is able to register only
the composite effect, it is possible to resolve this effect into its
component factors with the aid of a spectrophotometer. Although
the eye is unable to distinguish between a violet dye and a suitable
mixture of a red and a blue dye, the heterogeneous character of the
mixture is readily apparent upon spectrophotometric examination.
Pure dyes may have simple absorption spectra, in that their light
absorption is all at one part of the spectrum, or they may be more
complex, showing two or more points on the spectrum at each of
which light is absorbed to greater extent than on either side of it.
Thus even in the instance of pure products of identical color to the
eye, the spectrophotometer frequently reveals decided differences
when the character of the light absorption is considered in detail.

TABLE 1

SPECTRUM

(Showing Complementary Colors)

SPECTRUM

WAVE LENGTHS, m/x

COMPLEMENTARY COLORS

Violet

400-430

Yellow

Blue

430-490

Blue-green

490-510

Orange

Red

510-570

Green

Purple

Yellow

570-600

Violet
Blue

Orange

600-620

Blue-green

Red

620-750

While it may be necessary or advisable in some instances to
determine absorption in the infra red or ultra violet, the measure-
ment of absorption within the visible spectrum, upon which color
is dependent, is adequate for most purposes. The essential prin-
ciple of operation of a typical spectrophotometer is best under-
stood by considering first the principles of a visual instrument,
although such instruments are now practically obsolete because
of the greater efficiency of photoelectric measurements. These
principles may be understood by reference to Fig. 1. Such instru-
ments comprise essentially two beams of light from a common
source, one to pass through a glass cell containing the sample to
be examined, the other to pass through a similar cell containing

Spectrophotometric Analysis

55

solvent only; a photometer for adjusting the relative intensity of
the two beams; a spectrometer with prism and eye piece arranged
so that portions of the spectra of the light from the two beams are
observed juxtaposed in the field of the eye piece. The spectrum
of the beam which has passed through the dye solution will be
found deficient in those portions which have been absorbed by the
dye, and the degree of the deficiency at any position in the spec-
trum may be measured by determining the degree to which the
intensity of the light of the second spectrum must be reduced in

TABLE 2

RELATIONS BETWEEN THE SYMBOLS AND UNITS AND DEFINITIONS OF THE TERMS*

SYMBOLS

DEFINITIONS

A

1. Angstrom: also A; 1/6438.4696 of the wave length of the Cd red

line, or 10~*cm. = 0.1 m^t.

2. Absorption: 1 - T.

M

Micron = 10-*cm. = 1000 m/x = 10,000 A.

X

1. Wave length: expressed in terms of A or m/x (see 1. above).

2. Wave length of maximum absorption.

I

Transmitted intensity; light transmitted by solute.

lo

Incident intensity; light transmitted by solvent.

T

1. Transmission, transmittance and transmittancy: I/Iq.

2. Percentage transmission: 100 X I/Iq-

E

Extinction: also D or d; Logio Io/I> or ~Log T.

D

Density: also d or E; (see above)

c
d
k

e

Concentration of solution, gms. per liter.

Thickness of solution layer in cm. (Sometimes used to mean density).

Specific extinction coeflScient: E/cd.

Molecular extinction coefficient: Mk, (M = molecular weight).

I = lo- 10-kcd.

Ix)gio lo/I = kcd = E.

*For further discussion of this subject consult Erode (1943), Gibb (1942).

order to obtain an equal intensity in the two fields observed by the
eve.

The shutter of the eyepiece may be partially closed so that only
a narrow spectral range is visible ; this allows the eye to concentrate
on the matching of two small fields, each of which appears uniform
in color. The instrument is provided with a screw drum, calibrated
in wave lengths, by means of which the prism may be rotated in
such a manner as to bring light of any desired wave length into
the center of the field of vision.

56

Biological Stains

A

A ^i

^M

j£! P Ni

-^x

-tv

Z2^~> Slivered

Prism P

1
PHOTOlitTE-R-

PEC'TKOntTLR-

Fig. 1. Schematic diagram of Hilger-Nutting spectrophotometer. Specimen
holder with dye dissolved in a suitable solvent is placed at Ei while a similar cell
with solvent alone is placed at Ej. Both are equally illuminated from the same
light source. Through the partially silvered prism, P, the upper and lower portion of
the photometer field is illuminated through the specimen while the central portion is
illuminated through solvent alone. This latter illumination may be varied by the
rotation of the analyzer, Nj, which is actuated by a graduated circle C, reading with
the index I. By this means the illumination of the several portions of the field is
maintained equal and absorption values read off of circle C. The spectrometer of
the constant deviation type serves to isolate particular portions of the spectrum by
means of a graduated drum D which through a screw actuates the prism table sup-
porting prism B.

This visual type of instrument, as explained above, is now rarely
used, having become obsolete on account of the greater efficiency
of the photoelectric cell in comparison with the eye. The eye is
badly subject to fatigue after continuous use, and even at its best
has low sensitivity at the violet end of the spectrum. The photo-
electric cell, on the other hand, is not so subject to fatigue, can
make readings over a longer wavelength range, and even into the
ultra-violet, if special equipment for that purpose is provided.
For these reasons more rapid readings can be made, and it is pos-
sible to obtain the exact point of maximum absorption much more
quickly than with the eye. A photoelectric instrument such as
the General Electric Recording Spectrophotometer, is even more
automatic, as it has a motordriven device that records even the
most complicated absorption curve on a revolving drum in only a
few minutes. A recording spectrophotometer, however, is a rather
unnecessary refinement in the case of dyes, because of their rela-
tively simple absorption curves. Simple non-recording photo-
electric instruments, such as the Beckman or the Coleman, are
ordinarily suflBcient.

The optical principles of the Beckman are given in Fig. 2 and
a photograph of an assembled instrument in Fig. 3. Details of

Spectrophotometric Analysis

57

the Coleman instrument are not given; in principle it is quite
similar. In theory, such an instrument merely replaces the eye
with a photoelectric cell; but in actual practice the optical system
is different. This is due to the fact that the photoelectric cell
enables one to measure directly the intensity of the light by means

Fig. 2. Schematic optical diagram of Beckman model DU spectrophotometer

A.

Light source

F.

Quartz prism

B.

Condensing mirror

G.

Exit slit

C.

Diagonal mirror

I.

Absorption cell

D.

Entrance slit

K.

Photoelectric tube

E.

Collimating mirror

Fig. 3. Photograph of an assembled Beckman model DU spectrophotometer

of a potentiometer. With the eye as the recording agent, two
beams of light are necessary, one passing through the unknown,
the other through the solvent alone, plus an analyzer with which
the intensity of the light can be varied until the two beams of light
are equal in apparent strength. With the photoelectric cell, on

58 Biological Stains

the other hand, simultaneous inspection of two beams of light is
not necessary, nor is the analyzer for reducing the light intensity
needed; hence a single beam of light passing through the instru-
ment is all that is needed, with resulting greater simplicity of the
optical system. The chief bulk of a non-recording photoelectric
spectrophotometer is occupied by a potentiometer to record the
strength of the current generated by the photoelectric cell.

In measuring the complete visible absorption of a dye, a series
of measurements is made over the portion of the spectrum in
which any appreciable absorption may be noted. In doing this
with such an instrument as the Beckman, one of the absorption
cells is filled with the solvent alone, as a control, another with the
dye dissolved in the same solvent, in quite dilute solution. Then,
after checking the instrument as to its zero point the control cell
is placed in the beam of light coming from the exit slit, the mono-
chromator set to a point somewhat to one side of that part of the
spectrum where absorption is expected, and a reading taken as to
the intensity of the light reaching the phototube. Then a similar
reading is taken with the unknown sample. The setting is then
changed to a slightly different wavelength, and new readings taken.
In practice such readings are taken about every 5 m/x in the re-
gions of slight absorption but at least every 1 m/x near the peak.
The difference between the readings of the control and the un-
known at any point indicates the absorption of light at that
point. With the Beckman, four absorption tubes are provided,
which allows for readings to be obtained one after the other with
three different unknowns.

If measurements are carried out under suitable standardized
conditions, the spectral position and the general form of the ab-
sorption curve are characteristic of the individual dye, while the
magnitude of absorptive indices (the height of the curve) varies
directly with the amount of dye present. The absorption curves
of dyes which are very closely related in structure are sometimes so
similar as to be practically identical. In such instances the in-
dividual dyes may be recognized by means of quantitative deter-
minations of the degree in which their absorption is modified under
the influence of suitable variations in conditions.

The absorption curves of typical yellow, orange, red, violet, and
blue dyes are recorded in Figure 4. It will be noted that their maxi-
mum absorption in each case falls within the range of the comple-
mentary color (cf. Table 1). The great majority of dyes of these
colors, in the usual solvents and under the usual conditions, show
but one absorption band in the visible spectrum. The curves are
seldom perfectly symmetrical, however, and usually give indica-
tions of localized secondary absorption in some portion of the band.
It has been shown that this secondary absorption is due, in numer-
ous instances, to a tautomeric form of the dye. It should never be

Spectrophotometric Analysis

59

2.0

-

3.

—

1.9

-

j

1.8

-

\

4.

1.7

—

1

A

—

>* 1.6

-

j

/\

c

1 \

/ \

2 1.5

-

j 1

/ \

—

♦-

/ 1

/ \

i 1.4

(A

-

2.

/ I

/

5.

—

c

/ 1 >

f \

o 1.3

-

1/

\

*~

k.

jr

I

1-

/]

\

—

1.2

-

1

o

/

—

o> I.I

-

k /

'

o

\ 1

_l

\ 11

~

. '0

-

\ / 1

—

4-

\ /

«= 0.9

-

\ /

a>

1 1

\ /

,

u

A /

\ /

y

_

- 0.8

/\/

\ /

/

«^

^ V

1 /

/

• •

A

1 f

/

6 0.7

" A

1/

/

O

/\

1

/

^0.6

■/ '

A

/

o

/

/ \

\ /

• ^m

/

# I

1 /

_

*- 5

/

/ \

\ /

c

/ \

\ /

^ 0.4

1

' \

\/

K

\

y

UJ

\

X

1

0.3

' —

\ .

A

-

0.2

-

v

\

\ :

0.1
n

1

r 1 >

X ^

\

1 1 1

\ =

-0.01

- 0.02

- 0.03

- 0.04

0.05
0.06

0.07 **

- 0.08 =
o

0.09 1-

0.10 -

E

0.15 w

- 0.20

- 0.30

0.40
0.50
0.60

0.70

0.80

0.90

1.00
420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

Wave Lengths in Millimicrons

Fig. 4. Absorption curves of five dyes of different colors:

1. Tartrazine (yellow)

2. Orange G.

3. Fuchsin (red)

4. Crystal violet

5. Neptune blue BG.

accepted as evidence of the presence of a second dye unless it has
been ascertained that it is not found with a pure sample of the dye
under conditions of examination.

The absorption curve of a green dye is recorded in Fig. 5. It has

60

Biological Stains

420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

Wave Lengths in Millimicrons

Fig. 5. Absorption curve of malachite green.

a principal band in the orange and a secondary band in the violet.
Both the absorption curve and the color of the dye could be
matched closely by mixing a suitable blue and a yellow dye in the
correct proportions. All green dyes absorb appreciable amounts
of violet light as well as of red light.

Spedrophotometric Analysis

61

In Fig. 6 is given the absorption curve of a dye mixture, together
with the curves of the component dyes. The mixture is reported
to have been marketed in good faith as asymmetrical dimethyl
thionin, a dye which is intermediate in constitution and in color
between thionin and methylene blue (see under azure A, on p. 109).
The absorption curve plainly indicates the presence of two dyes,

C

0)

o

o

c
o

■*-
u

c

•*-

K

UJ

1.5

-

1.

2.

1.25

-

/ 1

A

1.0

-

/ 1

/ ^ I

0.75

-

/ //K

/ii

0.5

-

/ // 1

\

0.25

1 1 1

II 1 1 1 1

1 1 1 1

1.

2.
3.

440 460 480 500 520 540 560 580 600 620 640 660 680 700 720

Wave Lengths in Millinnicrons

Fig. 6. Absorption curves of:

Thionin.

Methylene blue.

Apparent mixture of these two dyes, incorrectly labelled (although in good

faith) a dye intermediate between them in chemical composition.

and suggests their probable identity. (It would be advisable to
effect the separation of small amounts of both dyes, if their positive
identification is desired.) The color of the mixture is very
similar to that of dimethyl thionin. The absorption curve of that
dye, however, is a simple and well-defined curve resembling those

62 . Biological Stains

of thionin and methylene blue, but occupying an intermediate
position in the spectrum.

This illustration shows how valuable the spectrophotometric
analysis may be in determining whether a given product is a simple
dye or a mixture of two or more dyes. This fact, together with its
use in determining the exact shade of any dye, makes it the most
valuable test to apply to a stain, other than to determine by actual
use whether the sample will prove satisfactory to the microscopist
or not.

The user of biological stains may well question, however,
whether spectrophotometric data have any actual value for him in
guiding him in the use of dyes. Such data as are given in this
book do in fact have a distinct use, provided he understands the
principle underlying them. Let us suppose, for example, that never
having heard of pyronin, he did not know its color; in such a case
the absorption maximum (552) as given on p. 178 would furnish
him this information. By referring to the spectrum and its compli-
mentary colors on page 54 he would find that an absorption maxi-
mum at 55^ means a primary absorption of green light; and as the
complementary color is red, he would thus learn that pyronin is a
red dye. In a similar way if he were trying to decide between using
one or the other of two closely related dyes, knowledge of their ab-
sorption maxima might help him, provided the decision depended
on shade. Thus if he were using azure A and obtained a shade
somewhat too red, the spectrophotometric data given on pp. 107-110
would show him whether to substitute azure B or azure C. It
will be seen that the absorption maxima of azures A, B, and C are
respectively 628, 652^ and 609. Reference again to the Table 1 on
p. 54 shows that the complementary color corresponding to wave-
length 65^ would be on the blue side of 628, while 609 would be on
the red side; accordingly azure B should be selected if bluer stain-
ing than with azure A were desired.

It should be remarked that the absorption curve of a dye is not
necessarily the same in two different solvents. In the following
pages aqueous solutions are to be understood unless some other
solvent is specified.

CHROMATOGRAPHY

Recently more and more stress is coming to be laid on a dif-
ferent method of dye analysis known as chromatography. It has
no real relation to spectrophotometry, but it is considered in this
chapter because it also furnishes a means of separation and iden-
tification of dyes on the basis of differences in color that are not
shown by simple visual comparison.

When two different chemicals are in solution together and are
brought into contact with porous substance in which capillary rise
readily takes place, it is rare that both compounds will pass through
the pores of the substance at the same rate. The result is the

Spedrophotometric Analysis 63

production of a coluiiiii in which each compound tends to be lo-
cated in definite bands. This phenomenon was naturally first
observed in the case of colored compounds; hence the name chroma-
tography, although it is now used, fully as much for uncolored as
for colored compounds. Dyes lend themselves naturally to this
method of analysis; although until recently it has not been much
used for them. The method is of value not only for showing the
various bands of color that develop, but actually to isolate the
individual constituents of the mixture, as the column can be cut
into its various sections and the chemical there localized can be
eluted by the proper reagent and thus obtained in pure form.

The method as originally developed called for a column of alum-
ina in a glass tube, in which the bands are located at points deter-
mined by the differing degrees of adsorption taking place between
the alumina and the different constituents of the mixture. A more
recent development, known as paper partition chromatography,
depends on a different principle, the relative solubility of different
compounds in two solvents, together with the aflSnity of cellulose
fibers for the solvents, which determines the rate of passage through
the pores of the filter paper. The paper partition method has
several advantages, among them the ease with which the chromato-
grams can be preserved and with which sections can be removed for
extraction of the individual fractions. Simultaneous papers
(Emery and Stotz, 1952; Christman and Trubey, 1952) have des-
cribed the application of the technic to dye analysis; and at about
the same time, Evans and Walls (1952) have shown the possi-
bility of getting very similar chromatographic pictures by the use
of filter paper electrophoresis.

A chromatographic strip obtained in this way from a mixture of
dyes can be very striking. Emery and Stotz show that a mixture
of three dyes (eosin Y, orange G, and erythrosin B) in a mixture
of butanol and water, and allowed to diffuse from a spot at the
top of a filter paper strip, develops five zones of color, one orange,
one red, two pink and fluorescent, one fluorescent without color in
ordinary illumination; of these five, the three fluorescent zones all
come from eosin Y, the other two from orange G and erythrosin
respectively. These authors, by cutting out the various zones
have been able to isolate the individual dyes and to figure the con-
centration of each in the original mixture.

This illustration is given merely to show the probable value of
the method. Much more work must be done, before it can be used
in routine dye analysis, and it is not likely to replace spectro-
photometry; but, as shown by Emery and Stotz it can serve to
reveal the identity of dyes in a mixture, even though one of them
(in this case eosin Y) is present in such strength as to mask the
others when analysis must depend on the spectrophotometer. Ac-
cordingly it is felt that chromatography may have an important
place in future assay methods for biological stains.

CHAPTER V

NITRO AND AZO DYES

1. NITROSO DYES (QUINONE OXIMES)

The nitroso dyes are produced by the action of nitrous acid on
phenoHc compounds, according to the equation

OH OH

J+HNO. = f |+H,0

NO

{nitrosophenol)

The fact, however, that the quinone oxime produced by the
action of hydroxylamine on p-quinone according to the equation

O O

l + NH.OH = f l+H.O

O NOH

(quinoneoxime)

is identical with the nitrosophenol produced as shown above,
shows that in all probability the substances can react as quinone-
oximes, which better accounts for their behavior as dyeing agents
since the quinone ring

or

Y ^'

occurs frequently in other dyes as a chromophore (see page 30).

as naphthol green y c. I. NO. 1*

Synonyms: Fast printing green. Gamhine.

NOH

v—O

C10H7NO2; Mol. Wt. 173.164
{An acid dye)

*This abbreviation stands for the number in the "Colour Index;" see Chapter
III, p. 36.

65

66 Biological Stains

a8 NAPHTHOL green B C. I. NO. 5

Synonyms: Naphthol green. Green PL. Acid green 0.
This dye probably has the formula:

NO-

Fe-

ON

NaOjS^

=^0 0=

30i]

C20H

loNsOioSzNaaFe;

Mol.

Wi. 604.260

{An

acid

dye)

Some naphthol green, probably the second of the above two
dyes, has recently been employed by Volkmann and Strauss (1934)
as a component of a tissue stain, the other dyes used in combina-
tion with it being gentian violet and azocarmine G. Mollier
(1938) calls for it in a quadruple tissue stain, with orcein, iron-
hematoxylin, and azocarmine G.

For the technic of MoUier's quadruple stain, see Staining Procedures*, p. IBs-T.

2. NITRO DYES

In this group the chromophore is -NO2. The chromophore is
of such a strongly acid character that the dyes of this group are
all acid dyes.

bS picric acid C. I. NO. J

Picric acid is formed by the action of nitric acid on phenol,
thus introducing three nitro groups:

OH

I

02N_/\_N02

Y

NO2

C6H3N3O7; Mol. Wt. 229.108

(An acid dye; absorption maximum about 360 in alcohol) \
Solubility at 26°C: in water 1.18%; in alcohol 8.96%

*Ccftin and Darrow (1943-4).

fWhen not otherwise specified, the absorption maximum is given for an aqueous
solution.

Nitro and Azo Dyes

67

This compound forms salts by the dissociation of the -OH group,
and the salts have considerable value as stains. Ammonium
picrate is most commonly thus used.

According to Dehn and Ball (1917), picric acid also has an
isomeric form

O O

II II
a,N /\=N— OH

NOj

This ortho-quinoid form occurs simultaneously with the benzoid
form, and changes in color of the compound may be explained by
disturbance of the equilibrium between the two isomers. The
quinoid isomer, with its -NOOH group, might well account for the
strongly acid character of the compound.

Picric acid (or one of its salts) is quite extensively employed in
contrast to acid fuchsin in the Van Gieson (1889) connective tissue
stain (which was originally proposed for staining nervous tissue).
It is also used as a general cytoplasmic stain in contrast to the
basic dyes. Wolf (1938) recommends it as a counters tain to
crystal violet in the cytology of fungi; Astbury and Preston (1940)
for staining cell walls of marine algae. It has further application
as a fixative for tissues that are to be sectioned and as a slow de-
calcifying agent.

PROCEDURES RECOMMENDED BY THE COM>nSSION IN WHICH THIS STAIN IS USED"

NAME OF PROCEDURE

PAGE REFERENCE

TO Staining Procedures

Van Gieson's stain with Iron hematoxylin
Picro anilin blue with Biebrich scarlet
Modified Gallego elastic tissue stain
Schmorl's method for bone sections
Picro anilin blue with safranin for woody sections
Goodpasture's stain for influenza bacilli in tissues
MacCallum's stain for influenza bacilli in tissues

IA4-2I

IB3-I2

IB3-I5

11)3-12

IIA-11

11163-15

IIIB3-U

*Under this heading are given references to procedures described in detail in
Staining Procedures, edited by Conn and Darrow (1943-4).

68 Biological Stains

Bio MARTIUS YELLOW C. I. NO. 9

Synonyms: Manchester yellow. Naphthol yellow.

This dye is usually the sodium, or sometimes calcium or am-
monium, salt of the following acid:

OH

I
. NO2

NO2
C10H6N2O5; Mol. Wt. 234.164

{An acid dye; absorption maxima about IflfB^ [399, 379] )

Solubility of sodium salt at 26° C: in water 1^.57%; in alcohol
0.16%. Solubility of calcium salt: in water 0.05%; in al-
cohol 1.90%)

Martins yellow has been used by Pianese (1896) in combination
with malachite green and acid fuchsin for studying cancer tissue;
the same technic was applied to plant tissue by Muller (1912), and
is now quite extensively used by plant pathologists in studying
sections of tissue infected by fungi. More recently it has been
employed by Nebel (1931) in contrast to resorcin blue for staining
pollen tubes in styles. Halbert (1935), by adding it to bacteriolog-
ical media, finds that it favors the growth of Escherichia coliy
but inhibits Aerobacter aerogenes. The dye is also used in prepar-
ing certain light filters used in photomicrography.

This dye is not at present available in America.

For technic of Pianese III B Stain, see Staining Procedures, p. IIA-12.

For technic of Lacmoid-martlus-yellow stain for pollen tubes, see Id. p. IIA-14.

Bl5 AURANTIA C. I. NO. 12.

Synonym: Imperial yellow.

This dye is the ammonium salt of hexanitro-diphenylamine.

NO. NOa O— NH,

O.N_/— \_N=/~\=N=0

V_/ \_/

/ /

NO. NO,

C12H8N8O12; Mol. Wt. 456.248

{An acid dye; absorption maximum about 4^25)

Solubility at 26°C: in water nil; in alcohol O.S3%o

Nitro and Azo Dyes 69

It is obsolete as a textile dye, although sometimes used as a photo-
graphic desensitizer. It is called for in combination with toluidine
blue and acid fuchsin in the Champy-Kull technic for demonstrat-
ing certain cell constituents (mitochondria, etc.) It is very
poisonous, frequently causing severe dermatitis.

Kassanis (1939) employs it in the KuU technic (i.e. with acid
fuchsin and toluidine blue) for the study of virus infested plants.
Favorsky (1939) uses it for inducing polyploidy in plants.

3. THE AZO GROUP

The azo dyes are characterized by the chromophore — N = N —
joining benzene or naphthalene rings, thus:

N = N

It is possible for the azo group to occur more than once in a mole-
cule, forming the disazo dyes, thus:

N=N

kA ,

N=N

The azo chrombphore is distinctly basic; but not sufficiently so to
make the dyes basic when they contain hydroxyl groups. Those
containing amino groups are, of course, pronouncedly basic.

The position of the hydroxyl or amino group on a benzene ring
in relation to the azo group is important. Ordinarily they are in
the para position to each other, thus :

(a

OH

The ortho position is next frequently assumed; rarely the meta
position. When the hydroxyl group assumes the ortho position the
character of the compound is quite distinct from that of the para
compounds. By a rearrangement of the atoms such a compound
is sure to change to a quinoid form, thus :

H O

A compound of this latter structure cannot form salts and does not
act as an ordinary dye. It does, however, prove to be soluble in

70 Biological Stains

oil and is able to color it by an apparently physical process. Hence
the azo-ortho-phenols, or azo-beta-naphthols, like Sudan III and
Sudon IV,

are important fat staining dyes.

B25 NUCLEAR FAST RED SALT R

OCH3

/\_N = N— ONa

N02-\/
C7H4N304Na; Mol. Wt. 217.123

This compound is not a dye, but is mentioned here because it
forms an azo dye when combined with phenols and certain phenolic
derivatives, and is used as a histochemical reagent, producing a
bright red in the tissue in the presence of such compounds. It is
best known to the biologist by the German name "Kernechtrotsalz
B" under which it has been sold by a stain company in that
country. Clara (1935) describes the use of such compounds for
this purpose. Jacobson (1939) similarly employs it in the study
of argentaflBne cells.

The compound is really a diazonium salt like those described in
Chapter X among the histochemical reagents. It is a diazo salt of
5-nitro-2-amino methoxybenzene closelv related to National Ani-
line's fast red salt B (see p. 234).

a. MoNO-Azo Dyes

Gl FAST YELLOW C. I. NO. 1 6

{Echt Gelh)
Synonyms: Fast yelloiv FY, G, S, BGy etc. Acid yelloui.

N = N SOsNa

NaSOa NH2

Ci2H9N306S2Na2; Mol. Wt. 401.330
{An acid dye; absorption maximum about It-90 in acid solution)
Solubility at 26° C: in water 18.^0%; in alcohol 0.24%.

Nitro and Azo Dyes 71

This dye is rarely used as a biological stain, but is called for by
Schaffer for staining sections of bone, and by Unna in certain stain
mixtures used in studying the phenomenon called by him chro-
molysis. Fast yellow G or GG is employed by Wallart and Hou-
ette (1934), together with hennatoxylin and acid fuchsin, in a
trichrome staining technic.

c5 OIL YELLOW II C. I. NO. 1 9

Synonyms : Butter yellow. Oil yellow D. Fast oil yellow B.

C14H15N3; MoL Wt. 225.284

Butter yellow is the most common designation for this dye in the
biological literature. The name is unsatisfactory for two reasons;
first, it sometimes falsely suggests some relationship to butter;
second, it is ambiguous because C. I. No. 17 (amino-azo-toluene)
is also called butter yellow. For these reasons the name given in
the above heading is preferred; or one may definitely specify it
by the chemical term, p-dimethyl-amino-azo-benzene.

It has not been used, apparently, as a stain, but has been con-
siderably studied as a carcinogenic substance, so much so that
fear has been expressed in some quarters that the public might
come to associate cancer with butter and that this food might be
thereby discredited.

c8 CHRYSOIDIN Y C. I. NO. lO

Synonyms: Brown salt R. Dark brown salt R.

N__N

C12H13N4CI; Mol. Wt. 248.713

{A basic dye; absorption maximum about ^61)

Solubility at 26°C: in water 0.86%; in alcohol 2.21%

This dye is a good substitute, in some procedures, for Bismarck
brown. Like the latter, it is easily injured by heat, and boiling
should be avoided in preparing solutions. Maheshwari and Wulff

(1937) have employed it in the vital staining of pollen tubes; Lison

(1938) for the vital staining of insects. Monn^ (1939) uses it to
demonstrate the Golgi apparatus. Varco and Visscher (1941) in-
clude it among a series of dyes employed in the study of gastric
secretion.

72 Biological Stains

cll BENZENE-AZO-a-NAPHTHYLAMINE

/\_N_N_/~"V_NH2

C16H13N3; Mol. Wt. 247.288
{Absorption maximum of hydrochloride 557.6 in alcohol)

A colored compound recently employed by Carter (1933) as a
vital stain for protozoa (probably in the form of its hydrochloride)
and given the abbreviated name "BAN". This product has ap-
parently never been put on the market as a commercial dye; only
its formula and chemical properties are known.

cl3 ORANGE G C. I. NO. ly

Synonym : Wool orhnge 2G.
Slightly different grade: Orange GG, GMP. Crystal orange GG.

SOsNa

/\N = N_ 1

\/ H0-| 1 ]

SOsNa
Ci6HioN207S2Na2; MoL Wt. 452.370

{An acid dye; absorption maximum about 476-4-80)
Solubility at 26°C: in water 10.86%; in alcohol 0.22%

This dye is strongly acid because of the two sulfonic groups.
It is one of the most valuable plasma stains in histological work.
It has great use as a background stain for hematoxylin and other
nuclear dyes in cytology. It is frequently employed, both by
botanists and zoologists, as a cytoplasmic stain, together with the
two nuclear dyes safranin and gentian violet in the Flemming triple
stain. It is of importance to the pathologist for its use with anilin
blue and acid fuchsin in the Mallory connective tissue stain; and
is used in various other double and triple staining methods, such
as that of Ehrlich-Biondi-Heidenhain, in which it is mixed with
methyl green and acid fuchsin. The Ehrlich "triacid mixture,"
also a combination of these same three dyes, is used in staining
blood. A further use is Bensley's "neutral gentian," a combi-
nation of orange G and gentian violet for staining the A and B cells
in the islands of Langerhans. An important recent application is
by Stoughton (1930), with thionin, for the differentiation of para-
sites in plant tissue.

Nitro and Azo Dyes

73

A spectrophotometric graph of this dye, compared with two
other orange dyes, is given in Fig. 7, page 74.

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH THIS STAIN IS USED

NAME OF PROCEDURE

PAGE REFERENCE

TO Staining Procedures

Mallory's anilin blue collagen stain

IB3-3

Heidenhain's "Azan" modification

IB3-4

Grossman's modification

IB3-5

Acid alizarin blue modification

IB3-6

Komhauser's "Quad" stain

IB3-8

Stoughton's thionin and orange G for fungi in tissue

IIA-12

Flemming's triple stain

IIB-10

Id., with iodine

IIB-11

Feulgen stain, De Tomasi modification

IIB-13

cl6

CHROMOTROPE 2.R

C. I. NO. 19

Synonyms: Chromotrope N2R. Chromotrope blue 2R. Fast
fuchsin G. XL Carmoisine 6R. Acid phloxine GR.

NaSOj SOiNa

Ci6HioN208S2Na2; Mol Wt. 468.370

{An acid dye; absorption maxima [5j^2] 503)
Solubility at 26°C: in water 19.3%; in alcohol 0.17%

This dye has recently been employed by Lendrum (1935) as a
counterstain to celestin blue in animal histology. Lillie (1940)
finds it is a fairly good substitute for "xylidine ponceau" in the
Masson (1929) trichrome stain. Crossmon (1940) uses it in the
selective staining of red blood cells in sections.

cl9 AZOPHLOXINE GA C. I. NO. 3 1

Synonyms: Fast crimson GR. Amidonaphthol red G.

OH NH COCH3

I I

N_N

NaSOs S03Na

Ci8Hi3N308S2Na2; Mol. Wt. 509.422
(An acid dye; absorption maxima 540 , 502)

74

Biological Stains

Very little mention has been made of this dye in the biological
literature but it has been called for by Dublin (1943) with "ponceau
de xylidine" as one step of the Bodian stain in neuropathology.

I 1

I I I

640

• J

700

WAVELENGTH m >*

Fig. 7. Spectral curves of three azo dyes:
1. Orange G. 2. Methyl orange. 3. Orange II.

c21

SORBINE RED

C. I. NO. 54

Synonyms: Azofuchsin SB. Kiton red S. Eriorubine G. Azo

acid red L. Azo rhodine 3G.

CHjCO NH.

OH

N_N_/\

NaSOs NaSOs

Ci8Hi3N308S2Na2; Mol. Wt. 509.422
{An acid dye: absorption maxima about [5^5], 4-99)

Nitro and Azo Dyes 75

Lillie (1940) suggests this dye (under the name Azofuchsin 3B)
among possible substitutes for "ponceau de xyhdene" in the
Masson trichrome technic.

AZO FUCHSIN

There is an occasional reference in histology to the use of an
azo fuchsin. Such references are not sufficiently definite, how-
ever, as there are several different azo fuchsins. The following,
for example, are recognized in the Colour Index:

Aso fuchsin 4G

C. I. No. 29

Azo fuchsin

C. I. No. 30

Azo fuchsin 3B

C. I. No. 54 (see p. 74)

Azo fuchsin 6B

C. I. No. 57

Azo fuchsin B

C. I. No. 66

Azo fuchsin G

C. I. No. 153

Azo fuchsin 6B,

GNorS

C. I. No. 154

All of these acid mono-azo dyes are closely related chemically
to orange G and Bordeaux red.

Of these, Lillie (1948) definitely specifies C. I. No. 153 as a con-
stituent of his naphthol blue black tissue stain.

c25 AZO ACID BLUE B C. I. NO. 59

There are various azo acid blues, with various shade designa-
tions attached to the names, but whose synonymy is somewhat
confusing.' They are mixtures; but an important constituent is
regarded as being

NaSOj
CisHisNaOoSXa; MoL Wt. 409.389

{An acid dye)

Menner (1935) has called for this dye in a stain for nervous
tissue. Otherwise there seem to be few, if any, references to it in
the biological literature.

76 Biological Stains

c«8 NITRAZINE

Synonyms : Nitrazine yellow. Delta dye indicator.

NO. OH

I

/\ N_N

NO. NaSOj SO3 Na

Ci6H8N40iiS2Na2; Mol. Wt. 542.370

{An acid dye)

This dye has recently been proposed as an indicator, having a
pH range of 6.0 to 7.2. Its acid color is bright yellow, from which
it passes thru gray-green into its alkaline color, bright blue.This
indicator can be used in the form of test strips, like litmus paper.
It is more sensitive than litmus, however, and shows greater color
contrast. Lillie (1940) finds it can replace xylidine ponceau in the
Masson (1929) trichrome stain.

For technic of Masson trichrome stain, Lillie modification, see Staining Pro-
cedures, p. IB3-I8.

cSl SUDAN II C. I. NO. 73

Synonyms: Oil scarlet. Fast oil orange II. Red B. Fat ponceau.

Orange RR.

C18H16N2O; Mol. Wt. 276.324

{An acid dye; absorption maxima in alcohol: 531. 5y 4.94-5)

Solubility at 26°C: in water nil; in alcohol 0.39%

In this dye the hydroxyl group is in the ortho position with re-
spect to the azo group. As explained above (p. 69), such com-
pounds show a tendency toward intramolecular rearrangement so
that the hydrogen atom detaches itself from the hydroxyl group
and becomes fixed to the neighboring nitrogen. Such a com-
pound is neither acid nor basic and, not being able to form salts, is
not an ordinary dye, but is fat soluble and has the power of color-
ing fat. Therefore, this compound, together with the following
and several others of similar structure described below, are fat
stains.

This fat stain has frequently been confused with oil red O be-
cause both are mistakenly given in the Colour Index as synonyms.

yUro and Azo Dyes 77

Lillie (1944c) has tried both dyes in his supersaturated isopropanol
technic, and finds Sudan II satisfactory, better in fact than Sudan
III, although not so intense or yielding such |a stable solution as oil
red O, oil red 4B or Sudan red 4B, it is especially good as a fat stain
for central nervous tissue, in combination with Weigert's technic
for myehn. (See p. 88.)

For Lillie's supersaturated isopropanol technic, see Staining Procedures, p.
ID3-20.

c35 PONCEAU 2.R C. I. NO. 79

Synonyms: Ponceau R, RG, G, 4^R, 2RE, XR, J, FR, GR.

Scarlet R. Xylidine ponceau 3RS. Lake ponceau. Brilliant

ponceau G. Sew ponceau Ji-R.

CH3 OH SOsNa

/ I /

H3C_/~\_N_N_/~\

/ \

~~SOgXa
Ci8Hi4N207S2Xa2; Mol. Wt. 480.422
{An acid dye; absorption maxima about \538\ 4-99)

A dye called xylidine ponceau, possibly this one, has been used
as an histological counterstain in the Masson technic (see Foot,
1933), and by others who have followed Masson's methods with
certain modifications. Lillie (1940), however, suggests other dyes
for this purpose which give better results. The exact identity of
the French "ponceau de xylidine" employed by Masson is not
known.

For the technic of Masson's trichrome stain, see Staining Procedures, p. IB3-I8.
c38 OIL BROWN D. C. I. NO. 8 1

Svnonvms: Sudan brown. Sudan brown AX. Fast oil brown S.
Brilliant fat brown B. Fat brown III.

N = N

OH
C20H14N2O; Mol. Wt. 298.328

Lillie (1944c) recommends Sudan brown, Sudan brown oB, and
oil brown D as satisfactory fat stains in the supersaturated iso-
propanol technic. These dyes all undoubtedly represent shades
of the above.

78 Biological Stains

c41 BORDEAUX RED C. I. NO. 88

Synonyms: Fast red B, BN or P. Cerasin R. Archelline 2B.
Azo- Bordeaux. Acid Bordeaux.

Various grades denoted: Bordeaux By BL, G, Ry R exira.

NaSOj SOjNa

C2oHi2N207S2Na2; Mol. Wt. 502.426

{An acid dye; absorption maximum about 520)

Solubility at 26° C: in water 3.88%; in alcohol 0.19%

Bordeaux red is used as a cytoplasmic stain, as, for example, in
aqueous solution when Heidenhain's hematoxylin is to be used
immediately afterward as a nuclear stain. It has also been used
with thionin and methyl green for staining sections, particularly
of spleen, testis, and liver. Lillie (1940) finds it a fairly satis-
factory substitute for xylidine ponceau in the Masson technic.

c45 SUDAN R C. I. NO. II3

Synonyms : Brilliant fat scarlet B. Oil vermillion.

OCH3 OH

C17H14N2O2; Md. Wt. 278.298

{A weakly acid dye; absorption maxima 569 \519.8])

Weichherz (1934) has recently used a Sudan R III (possibly this
dye or possibly Sudan III) in connection with the Kahn reaction
for the diagnosis of syphilis. It makes the reading of the tubes
easier.

c50 JANUS GREEN B C. I. NO. 133

Synonyms: Diazin green S. Union green B.

This is an azo dye having an azin as well as an azo chromophore
group, and is thus related to the safranins. It is a compound of
diethyl safranin with dimethyl anihn through an azo group.

Nitro and Azo Dyes

79

C2H

2"5

N

N

/

CH,

C2H

N / \ N^N

/ /\ CI

CH,

zns

r

CsoHsiNeCl; Mol. Wt. 511.053

{A basic dye; absorption maximum about 610-623)

Solubility at 26° C: in water 5.18%; in alcohol 1.12%

Janus green is best known for its use in demonstrating chondrio-
somes, stained intra vitam, according to the technic of Michaelis
(1900), and as more recently developed by Cowdry, and by Bens-
ley (1911). It is also used by Faris (1924) with neutral red for

I

I

I

I

bJ
O

3
8 •-•

400

• I I I I • I I ^ I I I

460 520 580 640
WAVELENGTH Tn>l

Fig. 8. Spectral curves of two azo dyes:
1. Janus greeru 2. Congo red.

I J

700

80 Biological Stains

sections of embryos, and has been employed in the vital staining
of fungi and protozoa; also the supravital staining of blood
(Sabin, 1929).

Soep (1927) proposes it in place of methylene blue for studying
reductase production in milk.

A spectrophotometric curve of this dye is given in graph 1, Fig.
8, p. 79. Like other green dyes, it has two absorption maxima,
one in the violet and the other near the orange range.

For use in Supravital staining of blood, see Staining Procedures, p. ID3-9.
For use in Lillie modification of Ziehl-Neelsen technic, see Id., p. IIIB3-II.

c52 JANUS BLACK C. I. NO. 134

Synonym: Diazine black.
CH3

/\/=N-\/\ /~\_0H

NH2 / \ N = N-\_/

CI /-\

C25H20N5OCI; Mol. Wt. 441.907
{A basic dye)

It will be seen from the above formula that this dye, like Janus
green B, is an azo-safranin. It has rarely been called for in biol-
ogy; but according to Maheshwari and Wulff (1937), Brestlavetz
has employed it in vital staining pollen tubes.

c68 METANIL YELLOW C. I. NO. 138

Synonyms: Orange MNO or MN. Acid yellow R. Soluble
yellow OL. Yellow M. Tropaeolin G.

NaSOa

N=N_/~\_NH_

CisHuNaOaSNa; MoL Wt. 375.373

{An acid dye; absorpiton maximum 536 in hydrochloric acid

solution)

Solubility at 26° C: in water 5.36%; in alcohol 1.45%

In Masson's (1929) technic (see Foot, 1933) this dye is used as a
connective tissue stain following hematoxylin and acid fuchsin.

For a technic adapted from Masson (1923), calling for Mucicarmine with hema-
toxylin and metanil yellow, see Staining Procedures, p. IA4-7.

Nitro and Azo Dyes 81

c60 METHYL ORANGE C. I. NO. I4i

Synonyms: Orange III. Helianthin. Gold orange MP.

Tropaeolin D.

N = N

/\/
NaSOa

Ci4Hi4N303SNa; Mol. Wt. 327.333

{A weakly acid dye; absorption maximum 506-507 in

acid solution)

Solubility at 26°C: in water 0.52%; in alcohol 0.08%

This dye has httle use as a stain, but is widely employed as an
indicator, as it is red in acid, and orange in alkaline solutions. Its
chief value as an indicator is that it is sensitive to mineral acids
without being affected by carbonates or most organic acids. It has
been used as an indicator for cell sap by Pfeffer (see Krause, p.
2305), and occasionally as a counterstain in histology. Under
the name of gold orange, it has been employed by Newcomer
(1938) as a counterstain to crystal violet in staining pollen tubes.

A spectral curve of this dye, compared with orange II and
orange G, is given in Fig. 7, p. 74.

c64 PRONTOSIL

Synonym : Prontosil red.

NH2SO2
C12H14N5O2SCI; Mol. Wt. 327.789

This is a red substance employed principally as a therapeutic
agent. It is essentially a basic dye in its chemical structure, al-
though not used as a dye. It is referred to here only because it has
been called for by Carter (1930) as a vital stain for insects and
plants.

82 Biological Stains

c68 ORANGE IV C. I. NO. 1 43

Synonyms: Orange N. Acid yellow D. Tropaeolin 00.

N=N

NaSOa NH

CisHiiNaOsSNa; Mol. Wt. 375.373

{An acid dye; absorption maximum about 527 in acid solution)

Solubility at 26''C: in water 0,16%; in alcohol 0.2%

The only biological use of this dye seems to be occasionally as
an indicator.

c7i BRILLIANT YELLOW S C. I. NO. 1 44

Synonyms: Curcumine. Yellow WR.

Ci8Hi3N306S2Na2; Mol. Wt. ^77.^22

{An acid dye)

This dye is closely related to metanil yellow and orange IV,
being manufactured by the sulfonation of the latter and containing
one more sulfonic acid group. The exact position of the latter in
the molecule is uncertain.

Margolena (1933b) has employed this dye in the Doglio (1932)
technic, in which it is employed as a counterstain to carbol fuchsin
in staining acid-fast bacteria. In Doglio's original paper the
exact nature of the dye used is not specified, however, and at least
two other dyes (C. I. Nos. 146 and 364) have been sold as brilliant
yellow.

c76 ORANGE I C. I. NO. I50

Synonyms: Naphthol orange. Tropaeolin G or 000 No. 1.

N = N

NaSOa

I
OH

Ci6HiiN204SNa; Mol. Wt. 350.321

{An acid dye; absorption maximum about ^76)

Solubility at 26°C: in water 5.17%o; in alcohol 0.64%)

Xitro and Azo Dyes 83

This is another dye which is turned red by excess of alkah and
has therefore some use as an indicator. McLean and Ireland
(1940) have employed it with malachite green for staining sections
of roots or stems of plants.

c76 ORANGE II C. I. NO. I51

Synonyms: Gold orange. Orange A, P, or R. Acid orange II ^
Y or A, Orange extra. Mandarin G. Tropaeolin 000 No. 2.

N = N

NaS03
Ci6HiiN204SNa; Mol. WL 350.321

{An acid dye; absorption maximum about 4SS-487)

Solubility at 26° C: in water 11.37%; in alcohol 0.15%

This dye, which differs from orange I only in the position of the
hydroxyl group on the naphthalene radical, is similar to it in color
and properties, but does not change color with changing reaction
of its solution. This dye is rarely employed in microscopic work,
and yet may be recommended as a valuable substitute for orange
G when a stronger yellow is desired for contrast purposes. Thus
French (1926b) uses it in combination with eosin and azure C in a
general tissue stain. It has been used by Bergonzini (1891) in
place of orange G in the Ehrlich-Biondi stain; and by Ebbinghaus
(1902) for staining keratin in sections of skin. Kalter (1943) in-
cludes it in a quadruple staining method, the other dyes in com-
bination with it being fast green FCF, safranin O and crystal
violet.

A spectral curve of this dye is given in graph 3, Fig. 7, p. 74.

c78 NARCEIN C. I. NO. 151

H OH

I

NaS03_/~^_N_N

\_/ I

NaSOg

C,6Hi2N207S2Na2; Mol. Wt. 454.386

{An acid dye)

Solubility at 26°C: in water 10.02%; in alcohol 0.06%)

This dye is a derivative of orange II, prepared from the latter
by treatment with sodium bisulfite. It is rarely used either as a

84 Biological Stains

textile dye or in microscopic technic. It has been called for by
Ehrlich, however, in combination with pyronin and methyl green
or methylene blue to form a neutral dye. It is no longer on the
market, however.

c81 AMARANTH C. I. NO. 1 84

Synonyms: Naphthol red S, C or 0. Fast red. Bordeaux.
Bordeaux SF. Victoria rubin 0. Azo rubin. Wool red.

N = N
HO \

NaSOs SOsNa

NaSOs

C2oHiiN20ioS3Na3; Mol. Wt. 604.475

{An acid dye; absorption maximum about 525)

Solubility at 26° C: in water 7.20%; in alcohol 0.01%

Amaranth is not a commonly used stain, but is of considerable
importance as a food color. It has been used by Griesbach (1886)
for staining axis cylinders, by Chambers (1935) in staining cells in
tissue culture; also by Smith (1939) in color photomicrography.

c85 METHYL RED C. I. NO. 2.11

COOH CHa

/
/~\_N=N/

CH3

C15H15N3O2; Mol. Wt. 269.294

{A weakly acid dye; absorption maximum 530)

This dye has rarely been used for staining, but has long been
employed as an indicator. Its useful range is from pH 4.4 to pH
6.0 in which it changes from red in acid solutions to yellow in
basic. Although still of value for this purpose it is coming to be
replaced by certain of the sulfonphthalein indicators such as brom
cresol green and chlor cresol green (see page 203), which are more
stable chemically and permit greater accuracy in reading. The
chief drawback to methyl red as an indicator is that it is easily
reduced with loss of color, and readings must be made very prompt-

Nitro and Azo Dyes 85

ly after adding it to the solution to avoid error due to this cause.
Carter (1933) has recently employed this dye in the vital staining
of protozoa.

Dl THIAZINE RED R C, I. NO. 12-5

Synonym : Chlorazol pink Y. Rosophenine lOB.

OH

N=: C_/~X_N = N_

/-v_ / \_/
\_/ s I

j SO3 Na

CH3 SO3 Na

C24Hi5N307S3Na2; Mol. Wt. 599.558

{An add dye; absorption maximum 505)

This dye has occasionally been used as a counterstain in animal
histology. It was introduced by M. Heidenhain (1903). It is
thought by some users to prove a satisfactory counterstain against
hematoxylin for skeletal and cardiac muscle.

b. Dis-Azo AND PoLY-Azo Dyes

d2 NAPHTHOL blue BLACK C. I. NO. 246

Synonyms: Pontacyl blue-black SX. Buffalo black NBR.

NH2 OH

I /
N02_/-^_N = N_/\/\_N =- N_/— \

\_/ J I i \-/

NaSOs SOsNa

Cr^HuNe O9 SaNaa; Mol. Wt. 616.505
{An add dye)

This dye has rarely been mentioned for biological use; but it
has been referred to by Lillie (1945c) as a stain for collagen. See
Staining Procedures p. IB3IO.

d3 brilliant CROCEINE C. I. NO. 252

Synonyms: Croceine scarlet 3By MOO.

NaSOa

/-\_N = N_/-

SOsNa

C22H13N4 O7 S2 Naa; Mol. Wt. 555.-^82
{An acid dye)

S6

Biological Stains

Brilliant croceine has been mentioned by Lillie (1945c) as a sub-
stitute for "xylidine ponceau" in the Masson triple stain.

cr
o

o
o

L I I I I I I I I l^\ I I I I J
400 460 520 580 640 700

WAVELENGTH Tn JH

Fig. 9. Spectral curves of three azo dyes:

1. Sudan III.

2. Sudan IV.

3. Sudan black B.

D4 SUDAN III C. I. NO. 148

Synonyms: Sudan G. Tony red. Scarlet B,fat soluble. Fat
ponceau G. Oil red AS, 0, B or SB. Cerasin red.

/\_N=N_

H0_

N = N"

C22H16N4O; Mol. Wt. 352.380
{A weakly acid dye; absorption maxima in alcohol 508-510)
Solubility at 26°C: in water nil; in alcohol 0.15%

Nitro and Azo Dyes 87

The chief value of this dye to the histologist is because of the
position of the hydroxy 1 group, which as explained above (pp. 69,
76) makes it oil soluble and hence a fat stain. It was introduced as
a fat stain by Daddi in 1896. Herxheimer (1901) investigated
this and several other fat stains, proposing formulae which are
still used as the basis of modern procedures. It is also employed
by botanists together with light green in the technic of Bugnon,
for differentiating suberized and cutinized tissue in plants.

For some time Sudan III was the only important fat stain
known. More is now known in regard to fat soluble stains, thanks
to the research of Michaelis (1901). It was he who showed the
relation of this property of certain dyes to their lack of basic or
acid character. He showed that new dyes with this property and
of greater staining power might be built up synthetically by taking
advantage of the fact that the azo group will attach itself in the
ortho position if the para position is already occupied. In this
way azo-ortho-phenols and beta-naphthols can be prepared, and
they prove to be fat soluble, Michaelis suggested the following
dye, which has now to a considerable extent replaced Sudan III.

Spectrophotometric graphs of Sudan III and Sudan IV are given
in Fig. 9, page 86.

d6 SUDAN IV C. I. NO. 2.58

Synonyms: Scarlet red.* Oil red IV. Fat ponceau.
Fat Ponceau R or LB. Cerotine ponceau SB.

C24H20N4O; Mol. Wt. 380.432

{A weakly add dye; absorption maxima in alcohol 522-529)

Solubility at 26°C: in water nil; in alcohol 0.09%

This di-azo naphthalene compound is similar to Sudan III ex-
cept that it is a dimethyl derivative. This fact makes it a deeper,
more intense stain; but having the hydroxyl group in the ortho

*Various erroneous variations of this term, some wholly or partly in German
(e.g. Scarlet R, Scharlach R, Scharlach red) occm- in the literatm-e and on labels.
They may be corruptions of the brand name, Biebrich scarlet R, Med.

88 Biological Stains

position, it has similar physical properties and is fat soluble. Until
recently it has been regarded as one of the best fat stains known;
see, however, oil red O and oil red 4B, below, oil blue NA (p. 228)
and Sudan black B, (p. 98).

For the technic of the Herxheimer stain for fat calling for the use of Sudan IV,
see Staining Procedures, p. ID3-I8. For Lillie's supersaturated isopropanol
technic, see p. ID3-2O.

d8 oil red O

As explained elsewhere, this dye has often been confused with
Sudan II. It is, however, quite different in composition, more
closely related to Sudan IV than to Sudan II. It consists of the
following together with its isomers having the methyl groups in
other positions:

CHs

N = N
/\/ \ CH

i /\/

CH3 CH3 N =

3

/
HO

\

C26H24N4O; Mol. Wt.

408.484

{A weakly add

dye)

Oil red O was recommended by French (1926c) as a substitute
for Sudan III, to be preferred because of its greater depth of color.
Lillie (1944c) recommends it for this same reason, regarding it as
one of the best fat stains known to him. It may be employed in
the Herxheimer formula (an approximately 0.1% solution in equal
parts of acetone and 70% alcohol), in the pyridin formula of
Proescher (1927), or in Lillie's supersaturated isopropanol technic.

For Proescher's oIl-red-0-pyridin technic, see Staining Procedures, p. ID3-I9.

For Lillie's supersaturated isopropanol technic, see Staining Procedures, p.
ID3-2O.

DlO OIL RED 4B

This is a dye of uncertain composition which is rarely referred
to in the biological literature, but mentioned by Lillie (1944c) as
one of the best fat stains to employ in the supersaturated iso-
propanol technic.

Nitro and Azo Dyes 89

Dl5 BIEBRICH SCARLET, WATER SOLUBLE C. I. NO. 2.8o

Synonyms: Croceine scarlet. Scarlet B, or EC. Ponceau B.

Double scarlet BSF.

C22Hi4N407S2Na2; Mol. Wt. 556.478
{An acid dye; absorption maximum about 503.5)
Solubility at 26°C: in alcohol 0.05%

The chief biological application of this dye is for medicinal pur-
poses, but it is occasionally used as a plasma stain, notably for
tissues after staining with polychrome methylene blue or Unna's
hematein. It has also been made use of by Paladino (1895) mixed
with alum hematoxylin for double staining effect on histological
material. In a neutral stain combination with ethyl violet, it has
been employed by Bowie (1924) for staining the islets of Langer-
hans. More recently it has been recommended by Lillie (1940) in
the Masson trichrome technic, by Schoor (1941) in combination
with orange G and fast green FCF for staining vaginal smears;
while McFarlane (1944) uses it in the modification of the Mallory
stain which he terms "Picro-Mallory".

For use of Biebrich scarlet with plcro-aniline-blue, or with methyl blue, see

Staining Procedures, p. IB3-I2.

For technic of Masson trichrome stain, Lillie modification, see Id. p. IB3-I8.

Dl6 PONCEAU S. C. I. XO. iSl

Synonym: Fast ponceau 2B.

OH

I
NaS03_/~X_N = N_/— \_N = N_/~\

\_/ \_/ v ■

CH3 \_

C23Hi7N404SNa; Mol. Wt. 468.282
{An acid dye)

Reference to this dye in biological literature shows a rather un-
usual history. It was mentioned quite early by Curtis (1905) who
found it an improvement over acid fuchsin in the \ anGieson stain.

90 Biological Stains

Little attention was paid to the suggestion, however, until quite
recently when the paper was discovered almost simultaneously by
Ruth (1946) and Leach (1946) both of whom found the Curtis
modification a useful one as it is less subject to fading.

D18 ORSEILLIN BB C. I. NO. 184

CHj

1 /

NaSO,

SOjNa
C24Hi8N407S2Na2; MoL Wt. 584.530
{An acid dye; absorption maximum 523)

Cohen and Doak (1935) employed this dye in contrast to crystal
violet for staining a mycorrhizal fungus on the roots of plants.
Subsequently other authors including Alcorn and Yeager (1937),
Keener (1951), have used it for staining other fungal parasites.
Maneval (1941) lists it among several useful bacterial stains.

d21 BISMARCK BROWN Y C. I. NO. 33 1

Synonyms: Vesuvin. Phenylene brown. Manchester brown.
Excelsior brown. Leather brown. Basic brown G, GX, or GXP.

Slightly different shade: Bismarck brown G.

(A basic dye; absorption maximum 463. Solubility at 26° C:
in water 1.36%; in alcohol 1.08%)

The various shades of Bismarck brown are mixtures of different
compounds, the most important of which are salts of the following:

N = N

/ \ NH2

/\ / \

NH2

N = N

\ NH2

/ \
\ /
NH2

The dihydrochloride is CisHzoNgCla; MoL Wt. 419.318

This dye was formerly employed quite extensively as a contrast
stain, but has now been replaced to some extent by others. It is

Nitro and Azo Dyes

91

still used, however, as a mucin stain, and is good for vital staining
and for staining in bulk. It is employed in staining cellulose walls
of plants in contrast to hematoxylin; and occasionally for staining
bacteria in contrast to gentian violet in the Gram technic, to
methyl or crystal violet in the Ljubinsky stain for diphtheria or-
ganisms, or to carbol-fuchsin for staining acid-fast bacteria.
Blaydes (1939) points out that it stains more intensely if used in
phenolic solution.

r I I I I n

H

-J
o

400

I I I I I I I ^?^^^ I I I
460 520 580 640
WAVELENGTH iTI^Ji

Fig. 10. Spectral curves of two azo dyes:
1. Bismarck brown Y. 2. Chlorazol black E.

I J
700

A caution to observe in connection with Bismarck brown is
that solutions should not be boiled before using, because the
composition of the dye is changed by heat.

A spectrophotometric curve of this dye is given in graph 1, Fig.
10.

For technic as mucin and cartilage stain in contrast to methyl green, see Staining
Procedures, p. IA4-5.

For technic of Ljubinsky stain for diphtheria, see Id, IIIA2-I2.

92 Biological Stains

d22 BISMARCK BROWN R C. I. NO. 332.

Synonyms: Bismarck brown GOOO 53 A or 53B. Brown R, AT,
C or N. Manchester brown EE. Vesuvin NR, B, R.

Basic brown BR or BXN.

C21H26N8CI2; Mol. Wt. 461.396
{A basic dye. . Solubility at 26° C: in water 1.10%, in alcohol 0.98%)

This dye has not ordinarily been employed as a biological stain.
One sample was submitted by a stain company by mistake, and
it appeared to be a satisfactory substitute for Bismarck brown Y.
Lillie (1945) specifies it as a good counterstain to oil blue NA.

d26 CONGO RED C. I. NO. 37O

Synonyms: Congo. Cotton red B or C. Direct red, C, R or Y.

I
NaSOi NaSOj

C32H22N606S2Na2; Mol. Wt. 696.658

(An acid dye; absorption maximum about 4-97)

Solubility at 26°C: in alcohol 0.19%)

This dye is best known to the biologist as an indicator. The
dye acid is blue, but its sodium salt is red. The red color of the
salt is readily changed by weak acids into blue. Besides serving
as an indicator, Congo red has certain histological uses, as for axis
cylinders (Griesbach, 1886), for embryo sections (Schaffer, 1888),
for staining elastic tissue (Matsuura, 1925), plant mucin, as a
stain for Uredineae (Blackman, 1905), and as a general background
stain in contrast to hematoxylin and other nuclear dyes. It has
been used by Klebs (1886) as a reagent for cellulose. It proves

Nitro and Azo Dyes 93

useful as a negative stain for bacteria (Benians, 1916, Cumley,
1935, Maneval, 1934) and for staining protozoa (Merton, 1932)
and yeasts (Gutstein, 1932).

A spectrophotometric curve of this dye is given in graph 2, Fig.
8, page 79.

For use of Congo red with Mayer's hemalum, see Staining Procedures, IA4-I3.
For use in Bennhold's stain for amyloid, see p. IA4-IO.

d31 ERIE GARNET B C. I. NO. 375

Synonyms: Congo corinth G or GW. Corinth brown G. Cotton

corinth G. Amanil garnet H. Direct garnet R. Buffalo garnet R.

Direct violet C. Diamine Bordeaux CGN.

NH. OH

I I

/\/V_N=N_/-X_/'^V_N=N _/\/\

'xa; ^/ ^/ yx;

NaSOj XaSOj

C32H2iN507S2Na2; Mol Wt. 697.642

{An acid dye)

This dye has been employed by Geschickter (1930b), mixed with
azure A, for staining frozen fresh tissue.

d35 trypan red C. I. NO. 438

SOaNa

NaSOj

NaSOi SOiNa

C32Hi9N60i5S5Na5; Mol. Wt. 1002.805
{An acid dye)

The chief use of this dye is as a vital stain. Varco and Vischer
(1941), for instance, thus use it in studies of gastric secretion. As
pointed out by AYhitby (1942) this dye has an interesting history
because it had very early application as a chemotherapeutic agent,
and led ultimately to the discovery of the arsenicals.

d38 BENZOPURPURIN 4B C. I. NO. 448

Synonyms: Cotton red Jf-B. Dianil red J^B. Diamin red J^B.

Sultan Jt.B. Direct red J^B.

94

Biological Stains

NH,

N = N /'

\_/

N=N

CH3

CH,

NaSOi NaSOi

C34H26N606S2Na2; Mol. Wt. 724.710

{An acid dye; absorption maximum about 497)
Solubility at S6°C: in alcohol 0.13%

This dye has been employed by Zschokke (1888) as a plasma
stain especially in contrast to hematoxylin, and by Ono (1934) for
staining spirochaetes.

d41 brilliant PURPURIN R C. I. NO. 454

NH,

./

BRILLIANT PURPURIN R

NH,

-/"

N=N

,/\ CH3 CH3 I

NaSOj NaSOj NaSOj

C34H25N609S3Na3; Mol. Wt. 826.759
{An acid dye)

Gutstein (1932) has employed a dye which he designates "bril-
liant purpur R" as a vital stain for yeasts. It probably is the
above. Lillie (1948) includes this dye, with azo fuchsin G and
naphthol blue black, in a connective tissue stain.

d45 vital red C. I. NO. 456

Synonyms: Brilliant Congo R. Brilliant Congo red R. Acid
Congo R. Azidine scarlet R. Brilliant vital red.

SOjNa

CH3

CH,

N_N

\

\/ \

H.N

x

SO.N:

r\=N / \

SOiNa

C34H25N609S3Na3; Mol. Wt. 826.759
{An acid dye; absorption maximum about ^98)

Nitro and Azo Dyes 95

This dye is a very important vital stain, much used by Evans.
(See Dawson, Evans and Whipple, 1920.) Commercially it is
better known under the name of brilliant Congo R. Difficulty
was at first found in securing a satisfactory product; but it was
overcome thanks to the cooperation of the Dept. of Agriculture,
and a good vital red is now obtainable from stain dealers.

d5o azo blue c. I. NO. 463

Synonym: Benzoin blue R. Direct violet B.

OH OH

N=:N_ _

■ /\

SO3 Na NaSOj

C34H24N408S2Na2; Mol. Wt. 726.678

{An acid dye; absorption maximum 560.5)

Monne (1935) has listed this dye among a series with which vital
staining of protozoa has been secured. Butt, Bonynge and Joyce
(1936) find that it can replace India ink in demonstrating capsules
of bacteria. Broda (1939) uses it in the histochemical demon-
stration of magnesium.

d52 DIANIL blue ZR C. I. NO. 465

Synonyms: Direct steel blue BB. Benzo new blue 2B.
Naphthamine brilliant blue 2R.

CH3

OH OH

N_N I I

NaSOj SOjNa

SOaNa

/

CH3

C34H23N40i2S3Na3; Mol Wt. 844.727
{An acid dye; absorption maximum about 568)

This has been used as a vital stain by Corner and Hurni (1918) ;
also by Sutter (1919), and Duran-Reynals (1937).

96 Biological Stains

D55 TRYPAN BLUE C. I. NO. 477

Synonyms: Chlorazol blue SB. Benzo blue SB. Dianil blue HSG.

Congo blue SB. Naphthamine blue SBX. Benzamine

blue SB. Azidine blue SB. Niagara blue SB.

H2N OH HO NH2

\ I N = N_/~V_/~X_N = N

CH CH

NaSOa SOaNa ' nIsOs SOsNa

C34H24N60i4S4Na4; Mol. Wt. 960.808
{An acid dye)

This is a valuable vital stain. By injection into the circulatory
system, it is employed for staining the'uriniferous tubules; also for
numerous other purposes in the vital staining of various kinds of
tissue, from mammals, lower vertebrates and insects.

d56 EVANS BLUE

OH NH2

V~\_/~\_N=N I /
\_/ \_/ \./\/\_S03Na

CH3 CHj ^^y

SOsNa SOsNa

C34H24N60i4S4Na4; Mol. Wt. 960.808

{An acid dye)

The similarity between Trypan blue and Evans blue is evident
by a glance at the two formulae. The latter, like the former, is
employed as a vital stain. It has been thus employed in several
rather varied applications: see Brunschwig, Schmitz and Jennings
(1940), and Zahl and Waters (1941).

d60 vital NEW RED

_NH CH,

N = N I \ O

\ I H II H |_/
-\N— C— N/
SOjNa NaS03

C35H28N807S2Na2; Mol. Wt. 782.752
{An acid dye)

No reference can be found to the manufacture of this dye except
in England. It is not listed in dye indexes, the dye most nearly

Nitro and Azo Dyes 97

like it thus recognized being C. 1. No. 353 (chlorazol fast pink
4BL) which differs from it primarily in that it is not methylated,
has two hydroxyl groups attached to the naphthalene rings, and
two sulfonic groups attached to the benzene rings. It has been
called for in certain vital staining procedures, having been intro-
duced for that purpose by Evans and Scott in 1921.

u65 NIAGARA BLUE 4B C. I. NO. 510

Synonyms : Pontamine sky blue 5BX. Direct sky blue.

Benzo sky blue.

H.N OH OH NH,

\ I N = N_/~\^/~\_N = N

./ \/\ OCH3 OCH3

NaS03 S03Na NaSOi SOjNa

C34H24N60i6S4Na4; Mol. Wt. 992.808
{An acid dye; absorption maxima about 6SJ!t,.2 and 589.5)

Occasional references have been made to this dye in connection
with histology for some time, although it has never come into
general use. Recently Varrelman (1938) has employed it for
demonstrating the vascular system of plants, taking advantage of
its rise through stems which are stood in a solution of the dye. In
the vital staining of skin or of tumors a pontamine sky blue has
been recommended, which may be this dye or may be C. I. No. 518
which has been sold under the name of pontamine sky blue
6BX (Syn. Niagara sky blue 6B or direct sky blue).

d68 MARSHALL RED

d70 HICKSON purple

These are two new dyes of unknown composition, but stated to
belong to the disazo group. They are discussed by Cannon (1941)
as having been manufactured in the course of his study of chlorazol
black E and of dyes of other colors found to be associated with that
dye. He claims that they are valuable nuclear stains in general
histology.

d75 chlorazol black E C. I. NO. 581

Synonyms: Pontamine black E, EX, or EXX. Erie black GXOO,

B, BF. Direct black MS, RL, E, GX, EE, 2V, F or A. Direct

deep black EW extra, E, EA, EAD extra. Renol black G.

98 Biological Stains

NH. HaN OH

_N = N_/\/%_N = N_/

NaSOj NaSOi

C34H25N907S2Na2; Mol. Wt. 781.726

{An acid dye; absorption maximum 598-602 in alcohol.)

This dye has only recently been introduced into microscopic
technic, Cannon (1937) having called attention to its use in gen-
eral histology and cytology. It proves to be a valuable stain
because it can be employed without mordanting or differentiation
and gives a very sharp clear-cut picture both of nuclear and
cytoplasmic structures. The black and gray tones it produces are
well suited for photography.

For use as general stain in animal histology, see Staining Procedures, p. 1A4-9,

For use as general stain in plant histology, see Id. p. IIA-6.

For use as plant cytologlcal stain with aceto-carmlne, see Id. p. IIB-4.

A spectral curve of this dye is given in graph 2, Fig. 10, p. 91.
It will be noticed that the black nature of the solution is indicated
by partial absorption through the whole visual range, with only
minor peaks at any wavelength.

d80 SUDAN BLACK B

_N = N _/~\_N = N _

C29H22N6; Mol. Wt. 554.414
{Absorption maximum 596-605.)

This compound is one of the most recently developed fat stains.
For a fat stain it has a rather unusual chemical structure, as it
does not show the typical -OH group in the ortho (or beta) position.
It was first prepared in Germany some time in the early '30's, and
became available in France and England. Lison and Dagnelie
(1935) proposed it as a myelin stain; but it was in England that its
value as a stain for fats was first developed (Gerard, 1935).
During the second World War it achieved general recognition for
the purpose, both in Great Britain and in the United States.
Hartman (1940) and later Burdon et at. (1942) found it valuable
as a stain for bacterial fat. It has also been proposed for staining
leucocyte granules and the Golgi apparatus.

Nitro and Azo Dyes 99

Unfortunately just at the time when its value was most ap-
preciated it went off the market entirely because of the war. It
was not till after the close of the war that American manufacturers
learned its formula and discovered how to make it.

A spectral curve of this dye is given in graph 3, Fig. 9, p. 86.
Characteristic of a black dye is the fact that there is considerable
light absorption even at the curve's minimum.

D82

(An acid dye)

Occasional reference has been made to an orange S in the biologi-
cal literature, which we are informed by the dye industry is prob-
ably of the above composition. It was proposed by DeGalantha
(1936) in a mixture with alum hematoxylin for staining connective
tissue and following Harris' hematoxylin for demonstrating
amyloid.

d85 victoria GREEI^ G

A new trisazo dye of unknown chemical composition studied by
Cannon (1941) in connection with other new azo dyes. He states
it to have valuable properties as a nuclear stain in general histology.

Other azo dyes sometimes mentioned in connection with hist-
ology are:

Janus red; C. I. No. 266.
Tropaeolin O; C.I. No. 148. Syn: Chrysoin. Gold yellow. Acme

yellow.

Tropaeolin Y; C. I. No. 148 (see note).

Roccellin; C. I. No. 176. Syn: Fast red A, AV, AL, BX, S or 0.
Cerasin. Rubidin. Cardinal red.

Crystal ponceau 6R; C. I. No. 89; S>ti: Ponceau 6R.

Carmine naphtha; C. I. No. 23. Syn: Sudan G. Oil yellow.

Alizarin yellow GG; C. I. No. 36. Syn: Anthracene yellow GG,
Mordant yellow 2GT.

Chlorazol paper brown B. A dye recently put out by the Im-
perial Chemical Industries of Manchester, England. Proposed
by Verdcourt (1947) for staining plant tissues.

Chrysoidin R; C. I. No. 21. '^ya: Cotton orange. Cerotin orange.

100

Biological Stains

Alizarin yellow R; C. I. No. 40. Syn: Alizarin orange. Mordant
yellow PN. Orange R. Anthracene yellow RN.

Diamond flavine G; C. I. No. 110.

Diamond black F; C. I. No. 299. Syn: Salicin black D. Chrome
black J or F.

Indole blue B or R; C. I. No. 135. Syn: Indoine blue BB, R or
BR. Safranin blue R. Janus blue G or R. Indophene blue.
Basilene fast blue B.

Direct violet K, N or R; C. I. No. 394. Syn: Chlorazol violet N.
Pontamine violet N. Diamine violet N. Erie violet 3R.

T

CHAPTER VI

THE QOXOXE-IMINE DYES

HE dyes of the quinone-imine group contain two chromo-
phore groups, the indamin group — N = , and the quinoid
benzene ring

They are derivatives of the theoretical compound paraquinone-
di-imine, which, if it existed in its free state, would have the
formula

HN

NH

In the typical indamin formula one of the imine hydrogen atoms is
replaced by a phenyl group, thus:

In the thiazins the introduction of a sulfur atom, attached to
both the phenyl and the quinone groups, forms a third closed ring.

as:

-NH

\/~N=\/
imino-thio-diphenylimine

In the oxazins an oxygen atom, and in the azins a nitrogen atom
takes the place of the sulfur of the thiazins, thus:

/X— NH

\/~N=X/
oxazin

H

I

\/~N=
azin

NH

1. THE INDAMINS

The indamin dyes are methylated amino derivatives of in-
damin. No dye in this group is a common biological stain. The

101

102 Biological Stains

following are occasionally mentioned, however, in connection with
histology:

h5 BINDSCHEDLEr's GREEN C. I. NO. 819

CH3 CH3

\ /

N_/~\_N=/~V=N— CH3

/ \_/ \_/ \

CHj CI

C16H20N3CI; MoL Wt. 289.801

{A basic dye; typically of the above formulay but sometimes sold as

the zinc double salt)

Bindschedler's green has occasional employment as an indicator
of oxidation reduction. It is reduced to the colorless pp'-tetra-
methyldiamino-diphenylamine. Elliott and Baker (1935) have re-
cently employed it for this purpose in studying the metabolism of
tumor tissue.

HlO TOLUYLENE BLUE C. I. NO. 8iO

NH, CHj

/ /

H2N_/"~\_N=/~\^N— C H3

\_/ \_/ \

/ CI

CH3

C15H19N4CI; MoL Wt. 290.791

{A basic dye)

Like its homolog, Bindschedler's green, toluylene blue is readily
reduced to a substituted diphenylamine; and accordingly it has
similar indicator properties.

2. THE INDOPHENOLS

Closely related to the indamines is a small group of colored com-
pounds known as indophenols, which do not readily form salts
with mineral acids. The simplest compound of this group is
indophenol (sometimes called benzenoneindophenol) and has the
following structural formula.

0_/~\_N /~\ OH

Various halogen substitution products of indophenol are known,
and all have indicator value, showing an acid-base color change
and a disappearance of color on reduction. The best known in-
dicator of this series is:

Quinone-imine Dyes 103

i5 -L, 6-DICHLOROINDOPHENOL

CI

I

=N_/^\_ONa ^ 0^/^\^N_/~X_OH

CI (blue) CI (red)

which on reduction becomes the following colorless compound

CI

HO /~\ NH /~^\ OH

It will be seen that the type of color change shown by this in-
dicator is very similar to that of litmus (see p. 217). Litmus is a
non-synthetic product of unknown chemical composition; it is not
believed to be related to the indophenols, in spite of its similar be-
havior as an acid-base and oxidation-reduction indicator.

There is only one other dye of this group that need be mentioned
here.

t20 indophenol blue c. I. NO .8ii

Synonym: Indophenol.

CII3

0-. ~ _ _, .

N

CH3

CisHieNzO; Mol. Wt. 276.324

{Absorption maximum in alcohol about 592)

This compound is occasionally referred to in biological work.
Herxheimer (1901), for example, employed a dye which he called
indophenol (and which probably was indophenol blue), in satu-
rated solution in 70% alcohol, as a fat stain. It is still sometimes
used for the same purpose; see Black (1938), for instance.

3. THE THIAZINS

The thiazins constitute one of the most important groups of
dyes from the standpoint of the biologist; while for textile dyeing
the group contains but a small number of dyes of any importance.
In these compounds, as mentioned above, the two benzene rings
are further joined by a sulfur atom.

Considerable information is at hand concerning the spectro-

104

Biological Stains

photometric characteristics of the thiazins (Knapp. Emery and
Stotz, 1950). From this information the absorption curves given
in Figs. 11 and 12 have been derived for seven of the dyes in the
group. Reference to these curves will be given under the indi-
vidual dyes as discussed below.

J5

THIONIN

Synonym: LautKs violet.

C. I. NO. 910

Thionin, having two amino groups, is a strongly basic dye. The
exact structural formulae of this dye and its derivatives, as well as
many others in which two benzene rings are similarly joined, are in
some dispute. At least two types of formulae are possible for the
thiazins and oxazins, as well as for the xanthene dyes (Chapter
VIII). One type is known as the orthoquinoid, the other as the
paraquinoid.

It is supposed (see p. 25) that when the quinoid ring is formed
the two hydrogen atoms replaced by atoms or groups v/ith double
valency bonds may be either in the para or in the ortho position to
each other. It will also be recalled from elementary chemistry
that sulfur and oxygen may be either bivalent or tetravalent. These
facts make it possible for a thiazin or an oxazin to have either one
or the other of the different structures represented by the following
two formulae for the theoretical thionin base:

HoN

.NH2

OH

"N^

paraquinoid fornmla

OH

I
H2N_/\_ S =/\_^NH2

orthoquinoid formula'

In the case of the paraquinoid formula the compound is an am-
monium base of the type discussed on p. 27, which is capable of
salt formation through its pentavalent nitrogen. In the case of the
orthoquinoid formula the salt formation takes place through the
tetravalent sulfur, the base being of the type known as a sulfonium
base. There are arguments in favor of either formula, and from
the standpoint of the biologist it does not matter which is pre-
ferred. Possibly both forms actually exist simultaneously. For

Quinone-imine Dyes

105

.9—

450

I t I

^90

I I I I I I I I I

530

570

I I I I I I I l-l
610 650 690

Fig. 11. Spectral curves of three thiazin dyes:
1. Methylene blue. 2. Thionin. 3. Methylene violet.

the sake of uniformity the paraquinoid form will be shown in the
following pages wherever possible; but with the understanding
that the orthoquinoid form is equally permissible.

106

Biological Stains

The dye, thionin, is a salt, generally a chloride, of the above-
mentioned base; and on the assumption of paraquinoid structure,
it has the following formula:

ILN

\/~N=\/
C12H10N3SCI; Mol. Wt. 263.741

{A basic dye; absorption maximum 598-599.)
Solubility at 26°C: in water 0.25%; in alcohol 0.25%

Thionin is violet in dilute solution, and has a very pronounced
metachromatic effect, the colors in sections stained with it ranging
from blue to reddish violet. A spectrophotometric graph of a
typical sample of thionin is given in Fig. 11, p. 105 (curve 2).

It is no longer used as a textile dye, and is to be distinguished
from thionin blue (C. I. No. 926) which is known to the trade and
has sometimes been furnished in place of the desired dye when
thionin has been ordered. Thionin is an especially valuable dye
for histological work on account of its metachromatic properties,
that is, its ability to impart different colors to different histological
or cytological structures. It is a very valuable chromatin and
mucin stain, proving especially useful in staining the tissue of
insects; and is recommended by Ehrlich because it stains amyloid
blue but mast cells and mucin red. It is a useful vital stain.
Perhaps its greatest value at the present time is in the staining of
frozen sections of fresh animal or human tissue, particularly in the
study of tumors. It is also used by Frost (1916) for staining very
young bacterial colonies in his "little plate" technic for counting
bacteria. (Unfortunately Frost specified thionin blue in one of his
papers, although the latter proves entirely unsatisfactory for the
purpose.)

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH THIS STAIN IS USED'

NAME OF PROCEDURE

Staining frozen sections

Staining fixed tissue

End point staining of Nissl granules

Schmorrs method for staining bone sections

Stoughton's technic (with orange G) for fungi in plant tissue

page reference to
Staining Procedures

IA4-I8
IA4-I9
IC3-U
ID3-I2
IIA-12

*Under this heading are given references to procedures described in detail in
Staining Procedures, edited by Conn and Darrow (194.3-52).

Quinone-imine Dyes 107

METHYLENE AZURE C. I. NO. 913

This was first recognized as one of the components of poly-
chrome methylene blue by Bemthsen (1885). It is now known
to be a mixture, not a simple dye. Definite knowledge of its
chemistry has been obtained by Kehrmann (1906), Bemthsen
(1906) and MacNeal (1906, 1925). It is regarded as primarily a
mixture of azure A and azure B, described below, compounds
which Bemthsen and MacNeal have shown simple methods for
preparing in relatively pure condition by the oxidation of methy-
lene blue. Another oxidation product of methylene blue, de-
scribed later by Holmes and French (1926), has been named azure
C. Now that these three dyes, azures A, B and C are recognized,
it would be most satisfactory if the name methylene azure could
be dropped, and users specify the actual dye employed.

Azure I (Giemsa) is a trade name applied to a secret preparation
which appears to be a somewhat variable mixture in which azure B
predominates. Azure II (Giemsa) is an intentional mixture of
azure I with an equal quantity of methylene blue. Products of
these same names are now sold by various companies and they are
certainly not all identical with the original Giemsa preparations.
They all probably agree, however, in containing azure A and azure
B as their chief constituents.

J6

AZURE

- c

This dye is mono-methyl thionin :

CHj

\

N

/
II

/\

s

/\

H

/
X— H

\
CI

C13H12X3SCI; MoL Wt. 277.767
{A basic dye; absorption maximum 607-610)

Curve 4 in Fig. 12, p. 108, is a spectrophotometric graph of a
typical sample of azure C.

It is not a textile dye; and up to the present has been manu-
factured only on a laboratory scale. A small lot, thus prepared,
has been put on sale by one of the American stain companies.
This dye, Hke the other azures described below, has up to the pres-
ent been prepared only by the oxidation of methylene blue. If
they prove sufficiently valuable, a process of manufacture by
direct synthesis will undoubtedly be developed. A product thus
manufactured would probably be cheaper as well as purer than the
present supply.

108
.6-

Biological Stains

0.4

L.I i I I I I I I I I I I I I I t I I
500 540 580 620 660

Fig. 12. Spectral curves of four thiazin dyes:

700

1. Toluidine blue.

2. Azure A.

3. Azure B.

4. Azure C.

Azure C is recommended by French (1926b) with orange II and
eosin as a tissue stain. Haynes (1926a) finds it possible to obtain
equally good results, however, with azure I (probably azure A) if
the technic is slightly modified. Later (1927) she reports excellent
staining with azure C; but it is still uncertain whether it gives re-
sults suflSciently different from thionin on the one hand and azure
A on the other to be of decided histological value.

Quinone-imine Dyes 109

J7 AZURE A

This Is now recognized as asymmetrical dimethyl thionin :

CH3 H

\ /

N_/\_S_/\ N— H

CH3

CI

Ci4Hi4N3SCl; Mol. Wt. 291.793
{A basic dye; absorption maximum 625-632)

Curve 2 in Fig. 12, p. 108, is a spectrophotometric graph of a
typical sample of Azure A. (Its similarity to toluidine blue is well
brought out in these curves.)

It is not a textile dye; but is probably present in much of the
methylene blue on the market. It must be distinguished from
the isomeric symmetrical compound :

CH3 CH3

\ , /

N_/\ S /\_N— H

"I
CI

/
H

which has never been given a special name. Ever since first recog-
nized by Kehrmann (1906) the impression has been current that
this latter dye has no staining value and is of a distinctly different
category from the azures. It is difficult to tell how this impression
has arisen, but possibly it may have been due to work with an
impure dye. Haynes (1927) found both isomers to have almost
identical staining properties.

There is no mistaking the value of azure A; it was in fact re-
garded by MacNeal (1925) as the most important nuclear staining
constituent of polychrome methylene blue. It was called for in the
originally proposed formulae for the tetrachrome blood stain (see p.
244). It has been certified by the Commission for some time.
Until recently it has been recommended for all purposes for which
methylene azure or Azure I w^as specified; recent work, however,
suggests that there are other constituents of the German Azure I
(particularly azure B) which may have special value in blood
stains.

Haynes (1926a) employed this dye as a nuclear stain preceding
eosin; and (1926b) following phloxine in a procedure similar to the
Mallory phloxine-methylene-blue technic. Douglas (1932) mixed
it with methylene blue for a counterstain to acid-fast bacteria.

For use in buffered azure-eosinate method see Staining Procedures, p. IA4-I7; in
Kinyoun's diphtheria stain, see p. IIIA2-I2.

110 Biological Stains

J9 AZURE B

Azure B is the tri-methyl derivative of thionin :

CH3 CH3

\ /

N_/\_S _/\_N-H

/ I I I I \

CH3 III CI

\/~N=\/

C15H16N3SCI; Mol. Wt. 305.819
{A basic dye; absorption maximum 648-653)

Like azure A, this compound was first recognized by Kehrmann
(1906) as a constituent of polychrome methylene blue. It can
easily be prepared in impure form by oxidation of methylene blue,
and is now available if anyone desires to obtain it.

Curve 3 in Fig. 12, p. 108, shows its spectral characteristics.
It is distinctly less red than any of the other azures.

MacNeal (1925) stated that its staining effects were much the
same as could be obtained with a mixture of azure A and methylene
blue, and that it therefore could be of little importance in poly-
chrome methylene blue. His statement has generally been inter-
preted as meaning that azure B is not a satisfactory stain. Holmes
and French (1926) also stated that this dye has little staining
value; but their conclusions were based upon work with an impure
sample, the performance of which was so poor as not apparently to
justify further purification. Haynes' later work (1927) on sections
of fixed tissue, with this same sample of azure B, agreed par-
tially with this, although she found the dye, if used in buffered
solution, rather better than fairly pure methylene blue in fresh
solution. Jordan and Heather (1929) definitely specify this dye in
a stain for Negri bodies. Moreover, recent tests by the Stain
Commission of certain exceptionally good Giemsa stains indicate
the possible presence of more azure B than in less satisfactory
batches. Roe, Lillie, and Wilcox (1940), in fact, have concluded
that azure B is one of the most important constituents of Giemsa
stain, especially when used for staining malaria parasites in thick
blood films. This work has been followed up by a series of other
papers by Lillie and his associates, and they have distributed
samples to other laboratories for testing; as a result it seems diffi-
cult to question their conclusions. Accordingly it is now recog-
nized that Azure B is one of the most important constituents of
''Azure I".

Quinone-imine Dyes 111

J 10 METHYLENE BLUE C. I. NO. 9^1

Synonym: Swiss blue.

Various grades denoted: Methylene blue BX, J5, BG, BB; grade
preferred for biological work: Methylene blue chloride.

Methylene blue is theoretically tetra-methyl thionin:

CH3

CH3

\ /

N /V_S /\_N— CH3

/

\

CHs

CI

C16H18N3SCI; Mol. Wt. 319.845
{A basic dye; absorption maximuniy if pure, about 66J^-666)

Solubility at 26° C: in water 3.55%; in alcohol l.Ji-8%

The dye is so readily oxidized that it is practically impossible to
obtain in pure form, the presence of some of the azures or of
methylene violet being almost universal. Methylene blue has
long been recognized as an important nuclear stain. It is notable,
however, that all the staining solutions recommended for use on
sections of fixed tissue call for some ripening or for the addition of
alkali. Such solutions are bound to contain one or more of the
azures. There are indications, in fact, that pure methylene blue is
a relatively poor histological stain.

A spectrophotometric graph of a reasonably pure sample of
methylene blue is given in curve 1, Fig. 11, p. 105. It is to be
noticed that this curve has a distinct "hump" at about 610 m/i;
this is characteristic of methylene blue and may indicate the pres-
ence of either a tautomeric form or of some product of oxidation.
It is interesting to observe that 610 is practically the absorption
maximum of azure C.

The methylene blue of commerce is generally a double salt, the
chloride of zinc and methylene blue. The zinc is toxic, however;
so for some time the zinc-free methylene blue chloride has been
prescribed for medicinal purposes, and is frequently designated
methylene blue Med. U. S. P. The zinc double salt is less soluble,
particularly in alcohol, so for most staining purposes is less desir-
able. The investigations of the Commission show that for all
ordinary staining purposes the zinc-free compound is best; so that
is the form at present recommended.

Methylene blue is perhaps the stain which the pathologist and
bacteriologist would have the greatest diflSculty in doing without,
and it is of great value to the zoologist as well. It is employed for
a greater variety of purposes than any other biological stain except

112 Biological Stains

possibly hematoxylin; and for this reason was the first dye to be
given a thorough investigation by the Commission. It is used:
first, as a nuclear stain in histology, for which purpose its strongly
basic character as well as the ease with which it can be applied
without over-staining, make it quite valuable; second, as a bacte-
rial stain, notably in milk work and in the diagnosis of diphtheria,
where it is especially useful because it has an aflSnity for the bac-
terial protoplasm as great as that of the rosanilin dyes, but is less
intense, more selective in its action and more subject to differen-
tiation; third, in the vital staining of nervous tissue, where a non-
toxic, basic dye is needed; fourth, in combination with eosin in the
blood stains, thanks to the ease with which it can be partly con-
verted into lower homologs such as the azures, and thus acquire
polychrome properties; fifth, as an oxidation-reduction indicator,
particularly in milk; and lastly, as an indicator in the Levine
eosin-methylene-blue medium for differentiating the colon and
aerogenes organisms.

It can be readily understood that an especially pure product is
needed when the dye is to be used for vital staining or in blood
work. For vital staining the U. S. P. zinc-free dye is always rec-
ommended, sometimes with even further purification ; although the
investigations carried on by the Commission indicate that the U.
S. P. product is sufficiently pure. For blood work some companies
sell a "methylene blue rectified for blood stains." This grade,
however, is generally less pure than the medicinal or U. S. P.
grade, and there seems no reason for specifying it. The same is
true of various other grades such as those denoted BX, BG, etc.,
which are ordinarily purer than the textile dye, but less pure than
the medicinal grade.

In former years, one or both of the German stain companies sold
a grade designated "methylene blue for bacilli". This was often
erroneously assumed to be specially adapted for staining bacteria.
As a matter of fact, it proved to be the zmc salt, quite impure, and
was probably labelled under the specified designation because it
was not considered good enough for any other purpose ! Even for
staining bacteria it did not prove especially satisfactory. (It is,
among other things, almost insoluble in alcohol, and cannot be
used in any formula — such as that of Loeffler — which is derived
from a stock alcoholic solution of the dye.) At present the Ameri-
can manufacturers, at least, have wisely discontinued the sale of
the zinc salt under this designation. One American concern,
however, still sells a "methylene blue for bacilli", but it is entirely
different from the former imported product of that name. It is
apparently methylene blue chloride, less pure than the medicinal
grade but entirely satisfactory for staining bacteria and for many
other staining purposes. The Commission has been unable, how-
ever, to find that it has any special advantages over the medicinal

Quinone-imine Dyes

113

product as a bacterial stain. It must be repeated that for all
staining purposes, except possibly some lines of vital staining,
medicinal methylene blue is an eminently satisfactory grade.
When ordered for staining purposes it is best specified under the
name: methylene blue chloride.

There is just one salt of methylene blue, other than the chloride,
which is recommended by the Commission — namely meihylene blue
thiocyanate. This form of the dye, however, is not recommended
as a stain but as an oxidation-reduction indicator for use in milk.
Thornton and Sandin (1935) have shown that this salt has distinct
advantages over the chloride in determining the rate of reduction
of milk by its bacterial flora; the thiocyanate contains no water
of hydration and hence can be obtained in almost 100% dye
strength — a fact which makes for more ready standardization of
solutions. Accordingly tablets of methylene blue for use in the
reduction test in milk, which are put on the market in America
are now wholly the thiocyanate.

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH THIS STAIN IS USED

NAME OF PROCEDURE

PAGE REFERENCE TO

Staining Procedures

Modified Wright stain for general tissue

IA4-I7

Tanzer-Unna orcein method for connective tissue

IB3-I6

Supravital staining of nerve endings (Ehrlich)

IC3-34

Wright stain

ID3-3

Tetrachrome stain, MacNeal

ID3-5

Staining smears of bone marrow

ID3-IO-II

Staining sections of bone marrow

ID3-II-I3

Counterstain to alizarin red S for calcium deposits

ID3-I6

General staining of bacterial smears

IIIA2-5

Counterstain in acid-fast staining

IIIA08

Staining the diphtheria organism

IIIA2-II

Macchiavello's stain for Rickettsiae

IIIA2-I9

Harris stain for Negri bodies

IIIA2-2O

Breed method for bacteria in milk smears

IIIA2-2I

Mallory's phloxine-methylene-blue stain

IA4-2O; IIIB3-4

Lillie modification of Ziehl-Neelsen technic

IIIB3-II

Kinyoun's diphtheria stain

IIIA2-I2

Stains for Negri bodies in sections

IIIB3-I2-I3

Polychrome methylene blue. The oxidation of methylene blue
into one or more of the compounds of lower methylation de-
scribed on the preceding pages takes place in any methylene bliie
solution upon standing, especially rapidly if the solution be al-
kaline as in the Loeffler formula. All methylene blue solutions
therefore, particularly if they have been standing any length of
time, contain small amounts of the lower homologs, primarily
azure A and azure B. These lower homologs are not only more

114 Biological Stains

violet in color than methylene blue itself but are more selective in
their staining action. It is indeed claimed (Scott and French,
1924b) that the dye owes much of its value as a nuclear stain to
the azures that are present, and that an extremely pure methylene
blue is not so satisfactory for such use. A later paper by Haynes
(1927) indicates the correctness of such conclusions.

A methylene blue containing noticeably large proportions of
the lower homologs is called polychrome methylene blue. The
formation of these oxidation products may be hastened by boiling
with alkali, as in Unna's formula.

Polychrome methylene blue is employed in many blood stains
such as Leishmann's and Wright's; in the latter a methylene
blue solution containing sodium bicarbonate is heated, and then
eosin is added. Eosin enters into chemical combination with the
basic dyes present, forming an insoluble compound which pre-
cipitates. This precipitate dissolved in methyl alcohol is Wright's
stain. (For a more detailed discussion of the subject see Chapter
XI).

Jl5 METHYLENE VIOLET (Bemthseu)

Methylene violet is formed whenever methylene blue is heated
with a fixed alkali or alkali carbonate. It is a feeble base with the
formula

C14H12N2OS; Mol. Wt. 256.312
{Absorption maximum 579-581)

Its preparation from methylene blue is more difficult than that of
azure A. A fair yield (30 to 40 per cent) may be obtained by
oxidizing methylene blue in dilute ammoniacal solution with
potassium chromate and then driving off the ammonia by boiling
with the addition of sodium carbonate. It may also be prepared
from azure A by boiling this with dilute alkali carbonate. Methy-
lene violet precipitates out as needle crystals, insoluble in water.
It may be recrystallized from dichlorethane (CH2CI • CH2CI) in
which it forms a deep carmine red solution. Although insoluble in
water when pure, methylene violet is soluble when mixed with
methylene blue or with the azures. It has a rather unusual shaped
absorption curve, as shown in graph 3, Fig. 11, p. 105.

It plays an important part in the nuclear and granule staining
of the polychrome methylene blue stains. A definite quantity of
this dye is employed in the tetrachrome Iblood stain of MacNeal.

Quinone-imine Dyes

115

For formula and technic of MacNeal's Tetrachrome Stain, see Staining Pro-
cedures, p. ID 3-5.

For use as counterstain to alizarin red S for calcium deposits, see Id. p. ID3-I6.

Methylene violet (Bernthsen 18$5) is not a textile dye and must
not be confused with methylene violet RRA or 3RA, which is
C. I. No. 842.

J20

CH3

CHa

\

I

/

N

METHYLENE GREEN

NO2 CHa

S_/\=N— CHa
CI

C. I. NO. 914

'N=

./

C16H17N4O2SCI; Mol. Wt. 364.845

{A basic dye; absorption maxima at about 660, 607.)

(Solubility at 26° C: in water 146%; in alcohol 0.12%)

This dye is a mono-nitro methylene blue, obtained by the action
of nitrous acid on methylene blue. The formula is probably as
given above, but the exact position of the nitro group is uncertain.

It is occasionally used as a substitute for methyl green, especially
by botanists in the case of wood and fixed chromatin, and gives
good results in combination with eosin. It has also been employed
by Lison (1938) as a vital stain for insects.

J25

TOLUIDINE BLUE O

C. I. NO. 915

Synonym : Methylene blue T 50 or T extra.

This dye is closely related to thionin and to methylene blue in
structure, and even more closely to azure A:

CHa

CHa

\

I

/

N_/\_ S_/X=NH2C1

CHa

C15H16N3SCI; Mol. Wt. 305.819

{A basic dye; absorption maximum 620-622.)

Solubility at 26°C: in water 3.82%); in alcohol 0.57%o

In practice it proves difficult to distinguish this dye from azure A
by spectrophotometric tests. This is particularly true because a
shift in the absorption band of toluidine blue O sometimes occurs in

116

Biological Stairis

storage as a result of which the two dyes may become almost
identical optically. Their great similarity is well brought out by
comparing Curve 1 (toluidine blue) and Curve 2 (Azure A) in
Fig. 12 on p. 108. Ball and Jackson (1953) have recently shown by
chromatography that toluidine blue O seems to be a mixture, and
suggest that this may account for much of the observed variation
between samples.

Although not a common textile dye, toluidine blue is more easily
prepared than thionin or azure A — a fact of considerable im-
portance, since in many procedures it may be substituted for one
or the other of these dyes. It may be employed like azure A as a
nuclear stain for sections of fixed tissue; and may be substituted
for thionin in staining frozen sections of fresh tissue. It is a valu-
able general nuclear stain, being ordinarily employed in 0.3-1%
aqueous solutions. It is widely employed in stains for Nissl gran-
ules and chromophylic bodies and has been proposed in a great
variety of special procedures. Well known among the latter is
Pappenheim's panchrome stain (of which it is an important in-
gredient) and the Albert stain which is at present widely replacing
methylene blue in the diagnosis of diphtheria. It has use as a
chemical reagent in the standardization of heparin and as a histo-
chemical reagent in determining thymonucleic acid.

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH THIS STAIN IS USED

NAME OF PROCEDURE

page reference to
Staining Procedures

Staining frozen sections
Bulk staining of Nissl granules
Alizarin red S method of staining bone
Albert's stain for diphtheria organism
Kinyoun's stain for diphtheria organism

IA4-I8-I9 (note)

IC3-I5

ID3-I5
IIIA.-ll
IIIA2-I2

j30

NEW METHYLENE BLUE N

Synonym: Methylene blue NN.

c. I. NO. 917

CH3"CH3

H

\

I

/

CH3

CH,

CH3CH,

N

'iN"

CI

C18H22N3SCI; Mol. Wt. 347.897

{A basic dye; absorption maxima about \636.Ii], 588.)

Solubility at 26° C: in water 13.32%; in alcohol 1.65%

Quinone-imine Dyes 117

This dye has practically never been called for in microscopical
work. The most interesting fact concerning it which has come to
light relates to the Van Wijhe technic as applied by Louise Smith
(1920) for staining the cartilage of frogs. The latter specified
methylene blue, but the results could not be duplicated with any
domestic or foreign methylene blue subsequently obtained. When
furnished through the Commission with samples of various stains
to try, she found that her earlier results could be duplicated with
new methylene blue — a fact which not only implies mislabeling of
her original supply of methylene blue, but suggests that new
methylene blue may have some value in histological work.

4. THE OXAZINS

This group is like the thiazins in chemical formula except that
the sulfur atom is replaced by an oxygen atom. Only a few of the
dyes find use in microscopic technic, and they are not stains having
very general application.

Ko BRILLIANT CRESYL BLUE C. I. NO. 877

Synonyms: Cresyl blue 2RN or BBS. Brilliant blue C.

CHj'CHz

\

N
/ \/X_0_/\_NH.
CH3-CH, I I I \

1 I 1 CI

CH3/\/-N==\/
C17H20N3OCI; Mol. Wt. 317.811

{A basic dye; absorption maxima about 62Jf.-6£8)

This dye is expensive to manufacture, and as it is not at present
employed in any line of commercial dyeing, it must be prepared
specially for the biologist. Its manufacturers, although willing to
cooperate to any reasonable extent, were naturally reluctant to
carry on much experimental work with this dye unless they were
sure the results would justify the outlay. This fact caused some
delay in bringing about its complete standardization.

The spectral curve of a typical sample of brilliant cresyl blue is
given in Fig. 13, p. 118, where it is compared with curves for resa-
zurin and Nile blue sulfate.

The dye has been employed to some extent in vital staining,
Irwin (1927), for instance, having found it the most favorable dye
available for investigating penetration into living cells; in vital

118 Biological Stains

o

•H .5-

o
o 4-

I 1

.3-

.2-

I-

I

z

UJ

o

QC
O

O Ci I I 1 i--h^| • I

^400 460 520 580 640 700

WAVELENGTH Tn }X

Fig. 13. Spectral curves of three oxazin dyes:
1. Brilliant cresyl blue. 2. Nile blue sulfate. 3. Resazurin.

staining it has special value because of its properties as an indicator
of oxidation-reduction. Of recent years it has been employed for
numerous purposes: staining protozoa, nematodes; in animal
cytology; and as an amebicide. Its best known use, however, is
as a blood stain to bring out reticulated cells and platelets. A well-
tried procedure is that of Cunningham (1920), as modified by
Isaacs {in McClung, 1937, p. 319); a somewhat different technic is
that of Robertson (1917) for counting reticulated cells, and that of
Buckman and Hallisey (1921) for platelets.

Of recent vears some interest has been aroused bv observations
that certain derivatives of brilliant cresyl blue stain tumor cells
selectively and retard the growth of tumors. See Lewis, Sloviter
and Goland (1946). The relation between ability to stain and to
retard growth is very interesting, even if not of practical signifi-
cance.

For technic of Cunningham's method of staining reticulated corpuscles and
platelets see Staining Procedures, p. ID3-8.

For technic of Robertsons' method for counting reticulated corpuscles, see Id.
p. ID3-8.

Quinone-iTnine Dyes 119

KlO GALLOCYANIN C. I. NO. 883

Synonyms: Alizarin blue RBN. Chrom blue GCB. Fast violet.

COOH

I
CH3 /\_N_

/
CH3

N~X/~0=\/"~0
OH

C15H12N2O5; Mol. Wt. 300.262
{Absorption maximum about 636)

Proescher and Arkush (1928) found the iron lake of this dye»
prepared by boiling 2-3 minutes in a 5% aqueous solution of ferric
ammonium sulfate, to be a splendid nuclear stain to be employed
as a substitute for hematoxylin. Buzaglo (1934) used it, in com-
bination with orcein and an acid alizarin blue, as a tissue stain.
It has also found employment (Foley, 1943) as a counterstain in
one of the protargol staining methods for nervous tissue.

For technic of Foley's combined method for staining nerve fibers, see Staining
Procedures, p. ICj-SS.

K15 GALLAMIN BLUE C. I. NO. 894

CONH2

CH3 /\_N_/\

\ I J II

N~\/~0=\/"~OH

/ I I

CH3 CI OH

C15H14N3O4CI; Mol. Wt. 335.743

(Absorption maximum about 651)

Employed in the form of its iron lake by Proescher and Arkush
(1928) as a nuclear stain; the lake is prepared as described for
gallocyanin above. It has been recommended by Stock (1949)
for detecting calcium in tissues.

The following very similar dye has also been mentioned in
biological literature, chiefly for its value as an oxidation-reduc-
tion indicator:

Prune pure; C. I. No. 893. Syn.: Violet PDH. Gallo blue E.
Very similar to gallamin blue, but a methyl ester instead of an
amide.

120 Biological Stains

k20 CELESTIN blue B C. I. NO. 90O

Synonym: Coreine 2R.

CONH2

I
C2H5 /\_N=/\

\ I I II

N~\/~0=\/~OH

/ i I

C2H5 CI OH

C17H18N3O4CI; MoL Wt. 363.795
{Absorption maxima about 654-5 [GOO])

Preferred by Proescher and Arkush (1928) to either gallocyanin
or gallamin blue as a nuclear stain. As in the case of these two
other dyes, the iron lake, prepared by boiling 2-3 minutes in a 5%
aqueous solution of ferric ammonium sulfate, is employed for the
staining solution. Lendrum and McFarlane (1940) recommend
this dye in a modification of the Mallory connective tissue stain.

k25 LACMOID C. I. NO. 908

Synonyms: Resorcin blue. Iris blue. Fluorescent blue.

There is some confusion as to the actual chemical structure of
this dye. It is recorded differently in different sources; and there
is little question but that it varies according to the manufacturer
from which it is obtained. At least one product of American
manufacturer is believed to have the following structure:

II II

NaO-\/\ /\/=0

O

This does not agree with the Colour Index, under the number
above cited, where a formula is given in which the NaO- radical
is replaced by NH4O-, and where a brominated derivative is re-
garded as properly called resorcin blue but not lacmoid.

Lacmoid has been used for its indicator properties. This dye,
apparently, was employed by Tsvett (1911) under the name of
resorcin blue, as a histochemical reagent for the detection of
callose. Nebel more recently (1931) combined it with martins
yellow (a dye at present unobtainable commercially) in a stain for
pollen tubes.

Quinone-imine Dyes 121

k30 NILE BLUE SULFATE C. I. NO. 913

Synonym : Nile blue A .

There are two products sold at present under this name, one is
a dimethyl aminophenol derivative, the other derived from diethyl
aminophenol. The formula of the latter is:

CH3'CH2

N
/ \/\_0_/\=NH2

N

(SO^)^

(C2oH2oN30)2S04; Mol. Wt. 732.828
{A basic dye; absorption maxima 63o-G4o)

A spectral graph of brilliant cresyl blue, compared with resa-
zurin and Nile blue sulfate, is given in Fig. 13, p. 118.

The use for which this dye is best known to the biologist is the
Lorrain Smith fat stain (Smith and Mair, 1911). In this pro-
cedure the dye is boiled with dilute sulfuric acid, and thus hydro-
lyzed, with the introduction of oxygen in the place of the radical
NH2 (S04)5, in other words producing a new dye of the class known
as oxazones. This oxazone dye is red, and is fat-soluble. Nile
blue sulfate itself, on the other hand, is not fat-soluble but com-
bines readily with fatty acids. As a result the technic serves to
distinguish between the free fatty acids in histological material
and the neutralized fats, the former staining blue, the latter red.
Knaysi (1941), however, calls attention to the fact that other dyes
(e.g. neutral red or methylene blue) can be similarly employed.

Nile blue sulfate is used unaltered for staining living tadpoles
previous to making transplants, in order to distinguish the grafts,
also as a supravital stain for embryos (Detwiler, 1917) and as a
vital stain for hydrae (Weimer, 1927), protozoa (Carter, 1933),
yeasts (Gutstein, 1932) and amphibian eggs (Wintrebert, 1932).

kland (1939) recommends it for staining bone sections, Tarao
(1940) for histochemical studies of the Golgi apparatus. Foshay
(1931) employs it for staining bacteria in tissues. The dye has
been employed in culture media, containing emulsified fat, for the
purpose of recognizing lipolytic bacteria; see Hammer and Collins
(1934). Loosanoff (1937) finds it useful in marking starfish for
identification.

For technic of Smith and Mair's stain for fats, see Staining Procedures, p. ID3-I8.

122 Biological Stains

k35 CRESYL violet

CRESYLECHT VIOLET (i.e. CRESYL FAST VIOLET)

The exact chemical nature of imported cresyl violet is not yet
known. Some samples appear to be mixtures of two dyes or
possibly two different forms of the same dye. Samples from
different sources frequently differ from one another but they are
all closely related dyes. Spectral curves of two foreign samples
are given in Fig. 14, graphs 2 & 3. They probably represent the
type of dye with which biologists were familiar in the days before
the world wars. Possibly, to distinguish them from the following,
the name "cresyl fast violet" should be retained for this foreign
product. It is not at present available.

At the present time a product of this name put on the market
by the National Aniline and Chemical Co. seems to be
different from any of the imported samples which have been exam-
ined. This National Aniline cresyl violet is better known chemi-
cally than the imported product and is considered to have the
formula:

CH3

\

N

/ \/\_0_/\_NH2
CH3 11 I I \

/\/-N=\/\ CI
CH3 I I

C19H18N3OCI; Mol. Wt. 339.815

{A basic dye; absorption maximum 62Jt.-630)

Solubility at 26°C: in water 0.38%; in alcohol 0.25%

A spectral curve of this dye is also included in Fig. 14 (graph 1) ;
a glance is sufficient to show how different it is from the foreign
product.

Like brilliant cresyl blue, this dye is not employed on a com-
mercial scale. Its chief value in histology is on account of its
metachromatic properties. It has been employed in making
permanent preparations of nervous tissue, and is excellent for
staining fresh areolar connective tissue, as it brings out various
histological elements. According to Ehrlich (1910, II, p. 78) it
stains nuclei violet, plasma blue, amyloid, mucin and mast cell
granules red. Spiridonovitch (1924) employs it in the vital stain-
ing of white blood cells. Williams (1923) uses it for staining sec-
tions of fresh tumor tissue in biopsy work. It is also employed
for making permanent preparations of fixed tumor tissue. Landau
(1934) proposes it for bulk staining of nervous tissue.

Quinone-imine Dyes 123

H r I I I.I -I

\

H.5-

Q 4-

CO

o

o

oLiiliiltiliiliiJ

400 460 520 580 640 700

WAVELENGTH m }i

Fig. 14. Spectral curves of cresyl violet.

1. National Aniline product.

2. Kresylecht violet from Bayer; 3. from Grubler.

There is evidence that a stain satisfactory for one of these two
latter purposes may not yield good results with the other. Wil-
liams, for example, finds rather better results with the National
Aniline product in biopsy work, while from other quarters com-
plaints have been received concerning the same batch of this dye
when used for making preparations from fixed tumor tissue, for
which the imported dye is very satisfactory.

There have been conflicting reports about the relative merits of
domestic and foreign products for neurological work. Quite un-
satisfactory results have been reported with the National Aniline
product as a stain for Nissl granules; but Banny and Clark (1950)
find that a cresyl violet just obtained from Coleman and Bell is
satisfactory. Their finding has not been verified by others. Fur-
thermore, the latter product is not the same as the foreign samples
shown by graphs 2 and 3 in Fig. 14. It is believed to be a diethyl
(instead of dimethyl) amino compound.

It is plain that further work is necessary both on the chemistry
of the imported product and on comparative value of the two
types in staining. Experimental work on this dye has not pro-

124 Biological Stains

gressed fast, both because of the small number of biologists who
employ it and because of the expense to manufacture; but it is
hoped that eventually it will be as well standardized as the more
commonly used stains.

k40 new blue R C. I. NO. 909

Synonyms: Naphthol blue R. Fast blue 3R. Phenylene blue.
Meldola's blue. Indin blue 2RD,

I I CH3

\/\_N_/\ /
1 I I UN-CH3

\/~o-\/ \

CI
C18H15N2OCI; Mol. Wt. 310.773
{A basic dye; absorption maxima about \622\, 573y \53S])

Occasional reference has been made to this dye in the biological
literature, under the name of naphthol blue R, most recently by
Lewis (1938) in staining fat drops in bacteria.

k45 RESAZURIN

O

H0~\/~"0"^/=0
C12H7NO4; Mol. Wt. 229.184

This is not a dye, but a colored compound that has interesting
properties as an oxidation-reduction indicator. It is blue in its
completely oxidized state, and upon reduction becomes pink and
then colorless according to the following reaction :

H

-\/""0^/=0 YiO~\/-~0~\/=0 HO"~\/~0-X/~OH
Resazurin Resorufin Hydroresorufin

(blue) (pink) (colorless)

Various shades between blue and light pink can occur.

A spectral curve of resazurin is included in Fig. 13, p. 118
(graph 3).

It was first suggested by Ramsdell et al. (1935) that it might be
more valuable than methylene blue as an indicator of reduction in
milk, partly because of its greater speed in showing reduction and

Quinone-imine Dyes

ns

partly because of the value of the intermediate pink stage which
does not occur with methylene blue. Its use was taken up quite
promptly in Great Britain, but more slowly in the United States.
It is now recognized as an alternate procedure by the Standard
Methods of the Amer. Public Health Assn., and standardized
tablets specially prepared for such use are on the market. For
recent papers on the subject see Johns and Howson (1940) and
Baker et al. (1942).

Another oxazin dye sometimes mentioned in connection with
histology is:

Capri Blue. C. I. No. 876.

5. THE AZINS

The dyes of the azin group are derivatives of phenazin, C6H4 -^^2 *
C6H4, a compound containing two benzene rings linked through
two nitrogen atoms in such a way as to form a third ring. Two
formulae are possible:

N_

X

and

N

In the case of the first formula the quinoid ring is the chromo-

— N—
phore; in the case of the second formula the azin group itself, I ,

_N—

(see page 29) is assumed to be the chromophore. The quinoid for-
mula is generally preferred today.

Phenazin is weakly basic, but is not a dye as it does not contain
auxochrome groups. In other words, it is a chromogen. Either
an -OH group or one or more -XH2 groups may be introduced to
give it dye properties. The acids and bases are very weak if there
is only one auxochrome group present, and their salts are readily
decomposed. For this reason some of them are of use as indi-
cators. Strong bases are encountered only among the safranins
where basic character is derived not only from the two -NH2
groups but also from one of the azin nitrogen atoms which be-
comes pentavalent and takes part in salt formation.

a. Amino-azins or Eurhodins

If one or more amino groups are introduced into a phenazin, a
dye is formed of the class known as eurhodins. They are very weak
bases, and therefore weak dyes; but as their salts are readily de-
composed with a resulting color change, they form useful indi-
cators. The best known of the group is toluylene red, base:

126

Biological Stains

\

CH,

/

N_/\_N_

NH2
_CH3

The chloride of toluylene red is the well-known neutral red.

l5

NEUTRAL RED

C. I. NO. 8x5

Synonym : Toluylene red
CH3

CHj

/

CI H

N_/\_N_/\_N— H

\
H

CH3
C15H17N4CI; Mol. Wt. 288.775

{A weakly basic dye; absorption maximum 54-0-54^)
Solubility at 26° C: in water 5.64%; in alcohol 2.45%

This dye is yellow in solutions more basic than the neutral point
(i.e., pH = 7.0) in reaction, and red in weak acids, even the re-
action of ordinary tap water being sufficient to bring out the
acid color; at a higher range of acid it turns blue. This gives it
some value as an indicator. As an indicator it is also used in
bacteriological media for distinguishing the colon from the typhoid
organisms, and for recognizing other forms; although it is employed
for this purpose much less today now that other dyes have been
shown to have even greater value for the same type of work.

A spectral curve of this dye is included in Fig. 15, p. 127, to-
gether with one of azocarmine G.

Neutral red is employed in a variety of staining methods. It is
a weak nuclear stain, and has value for that purpose in certain
procedures. Twort's (1924) stain for parasites in tissues is a
neutral stain (see Chapter VIII) formed by combining neutral red
with light green and dissolving the precipitate in alcohol. Neutral
red finds use in the study of the Golgi apparatus in cells; but there
is a dispute as to whether it is actually a specific stain for this
structure or merely for the inclusions of the Golgi apparatus. Old
ripened solutions are employed for bringing out the Nissl granules
in nerve cells. It also has some use in general histological stain-
ing, particularly for embryological tissue in combination with
Janus green, as recommended by Faris (1924). Knaysi (1941)
points out that it can be employed in place of Nile blue A in dem-
onstrating hydrolysis of fats.

Quinone-imine Dyes 127

O tTi I I I I I I I I rNo I I I J
*'400 460 520 580 640 700

WAVELENGTH Tn >x

Fig. 15. Spectral curves of two azin dyes:
1. Neutral red. 2. Azocarmine G.

It has special value where a weakly basic, non-toxic dye is
called for, as in vital staining. It is used for staining living pro-
tozoa, and as a vital stain for nuclei in tissue; also for the 'Vital"
staining of blood, that is of fresh blood, observed under a micro-
scope in a moist chamber, and for staining fresh gonorrhoeal pus
under similar conditions. It proves useful as an indicator of re-
action of the contents of living plant cells. The chief draw-back to
neutral red in vital staining is the toxicity of certain lots that have
been on the market. This toxicity seems to be due to impurities
present in the dye. Phillips and Cohen (1927) have, in fact,
shown that these impurities can be eliminated more readily by
preparing the dye as an iodide instead of a chloride. Their process,
however, has not been adopted by manufacturers as yet, because
of technical difficulties. Nevertheless the recent samples of neu-
tral red chloride submitted for certification have been so well
purified that they seem to be free from toxicity.

For technic of supravital staining of blood, with Janus green, see Staining Pro-
cedures, p. IDa-9.

128

Biological Stains

l8

NEUTRAL VIOLET

CH3

\

C. I. NO. 82.6
CH3

N

N.

NH.HCl

N

CHj J CIIj

\/-N=\y NH-\/

C22H25N6CI; Mol. Wt. 408.925

{A weakly basic dye; absorption maximum 533)

Solubility at 26°C: in water 3.27%; in alcohol 2.22%

This dye is very similar in its properties to neutral red, except
that, due to its greater molecular weight, it is more bluish, giving
a violet instead of a red color. It can be used as an indicator, but
has been seldom used in histology. Unna (1921) however, has
used it in a dye mixture employed in the study of chromolysis.

b. Safranins

Quite a long series of azin dyes are known in which one of the
nitrogen atoms of the azin group is pentavalent and another ben-
zene ring is attached to it. This pentavalent nitrogen allows the
compounds to behave like ammonium bases; so with the amino
groups which are always present, the basic properties of these dyes
are very strong. The theoretical base of the simplest safranin
would have the formula:

HaN

This form of ammonium base does not actually exist, as the safra-
nin bases really occur in the form of anhydrides; but salts of these
ammonium bases are the commonly known dyes. The com-
mercial dyes are ordinarily chlorides.

There are two groups of safranins : the benzo-safranins in which
the azin group unites two benzene rings; and the naphtho-safranins
in which it unites two naphthalene groups. The simplest safranin
is pheno-safranin, which is the chloride of the theoretical base just
given, namely:

PHENO-SAFRANIN C. I. NO. 840

Synonym : Safranin B extra.

/\ N_/\

Lis

H,N / \ NH,

/\ CI

C18H15N4CI; Mol. Wt. 322.789

Quinone-imine Dyes 129

Moore (1933) has called for phenosafranin in staining colonics of
bacteria and fungi. The commercial safranins are ordinarily
methyl or ethyl substitution products of this; or occasionally
phenyl substitution products. The one of greatest value to the
biologist is generally called safranin O.

L20 SAFRANIN O C. I. NO. 84 1

Synonyms : Safranin Y or A. Gossypimine. Cotton red.

SHghtly different shades: Safranin AG, T, MP, and G. (Although
all included in C. I. No. 841 they are different from the grade
here described.)

{A basic dye; absorption maximum about 530)
Solubility at 26° C: in water 5.4.5%; in alcohol 3.41%

The common safranins of commerce, under various shade
designations, are mixtures of di-methyl and tri-methyl pheno-

C2oHi«N4Cl; Mol. Wt. 350.841 C21H21N4CI; Mol. Wt. 364.867

The shade differs according to the proportion of these compounds
present, the red being deeper according to the proportion of the
tri-methyl compound in the mixture. The type safranin O, which
proves best for ordinary biological purposes, can be defined as
having its absorption maximum at 530m /x.

A spectral curve of safranin O is given in Fig. 16.

Safranin has always been a problem, because of its variability,
especially in cytological work where it is often employed together
with another basic dye, and a delicate balance between the two
stains is necessary. The reason for this variation has been a
puzzle ever since the Commission has begun its investigations of
this dye. The question is not yet answered. It is realized at
present, however, that there are samples that stain too blue, and
others that stain too yellow, although this difference does not cor-
relate with dye content nor with any chemical properties of the
dye yet investigated. Strangest to say, there is no difference be-
tween any of these types of safranin that can be recognized by the
spectrophotometer. Under such circumstances, no truly scientific
method of standardizing the stain has been|devised. An empirical
method is in effect, however, which results in the present product

130 Biological Stains

rill

.6-

M

\

o

•-♦ ,5-

ID

O .4-

.3-

en
^ 2-

o

y 1

—

o

400

i 1 1 1 1 1 1 (^ 1 1 1

1 J

460 520 560 640

700

WAVELENGTH Tnu

Fig. 16. Spectral curve of safranin 0.

being distinctly better than that of former years. When a stain
company submits a sample showing too blue or too yellow staining,
the fault is called to the manufacturer's attention, and they try to
submit another sample without that fault or with the opposite
fault. This often means the submission of two batches, one too
blue in its staining properties, the other too yellow; then it is a
fairly simple matter to determine the proper mixture of the two to
give best results. This has to be determined by actual staining
tests, not by means of any colorimeter or spectrophotometer.

These observations undoubtedly explain the directions given by
Chamberlain* to mix equal parts of alcohol soluble safranin and
water soluble safranin for use in the Flemming triple stain. There
is no such thing as a safranin insoluble in either alcohol or water;
and the products formerly sold by Grubler as "wasserloslich" and
"spritloslich" respectively have been found to be identical so far
as chemical and optical analysis show. Probably, however, the
particular batches formerly employed by Chamberlain were
neither of them the correct shade in staining properties, while a

*In early editions of his Methods in Plant Histology.

Quinone-imine Dyes

131

mixture of the two gave good results. In other words he did about
the same thing that the Commission now often recommends stain
companies to do.

Grlibler also sold other grades of safranin, none of which seem to
have any special significance for biological purposes. One of these
denoted "Safranin pur" was found on testing to be a more violet
dye, apparently methylene violet (C. I. No. 842).

Safranin O is one of the most important nuclear stains known to
the histologist. The botanist finds it especially valuable, as it
brings out lignified and cutinized tissues in vascular plants, and
can be employed in combination with a variety of contrast stains;
it is valuable as a protein stain in plants, and can be used to stain
spore coats. The cytologist makes use of it in the Benda technic
to stain chromatin in combination with light green as a contrast
stain; and even more widely in the Flemming triple stain, in which
it is employed as a chromatin stain, together with gentian violet
and orange G. The bacteriologist has some use for it, especially
as a counterstain in the Gram technic.

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH THIS STAIN IS USED

PAGE REFERENCE TO

NAME OF PROCEDURE

Staining Procedures

Counterstain to Welgert's iron hematoxylin

IA4-6

Counterstain to AgNOs for calcium deposits

ID3-I7

Counterstain to iron alum hematoxylin in plant histology

IIA-9

With light green SF yellowish in plant histology

IIA-10

With picro-anilin-blue in plant histology

IIA-11

In Flemming's triple stain

IIB-10, 11

With fast green FCF in plant cytology

IIB-11

With anilin blue WS in plant cytology

IIB-12

Counterstain to Foster's tannic acid and FeCls

nB-12

Counterstain in Gram technic, for smears

IIIA2- 6

Schaeffer and Fulton spore stain for bacteria

IIIA2-IO

Counterstain in Gram technic, for sections

IIIB3-9

l25 amethyst violet

Synonyms: Heliotrope B. Iris violet.
This dye is tetra-ethyl pheno-saf ranin :

CH3CH. 11 I I CH.CH,

\ /\/~N=\/\ /

N / \ N

/ /\ CI \

CH3CH. 1 I CH.CHj

C. I. NO. 847

C26H31N4CI; Mol. Wt. 434.997
{A basic dye; absorption maxima about 589, [54-5.5])
Solubility at 26°C: in water 3.12%; in alcohol 3.66%

132 Biological Stains

Amethyst violet has been used by EhrHch and Lazarus (1898) as a
basic dye in certain triple staining technics.

A further dye of this group which the biologist must take into
account, although probably of no significance as a stain, is methy-
lene violet RRA or 3RA, C.I. No. 842 (syn. : fuchsia or safranin
extra bluish) . This dye is a di-methyl safranin in which the methyl
groups are introduced into one of the amino groups instead of
directly into the benzene ring. It has no connection with the
methylene violet of Bernthsen, which is one of the constituents of
polychrome methylene blue; see p. 114. A sample has been found
labelled ''Safranin, pur", Grubler.

l30 azocarmine g c. I. NO. 8i8

Synonyms: Azocarmine GX. Rosazine. Rosinduline GXF.
The sodium salt of a disulfonic acid of phenylrosinduline :

C28H20N3CI; Mol. Wt. 433.921
{An acid dye; absorption maxima about 512)

A spectral curve for this dye, together with one for neutral red,
is given in Fig. 15, p. 127.

This dye is occasionally employed in tissue staining, notably
after M. Heidenhain (1915) who used it as a tissue stain either
alone or preceding some nuclear stain. Heidenhain gave sj>ecial
attention to its use in the Mallory anilin blue connective tissue
stain; in which procedure he employed it in place of acid fuchsin
in the Mallory technic. This azocarmine modification of the
Mallory stain is very commonly followed according to the schedule
given by McGregor (1929) which calls for staining in azocarmine,
differentiation in anilin alcohol, then treatment in 5% phospho-
tungstic acid before application of the Mallory anilin-blue-orange-
G mixture. (Unfortunately McGregor calls the dye ''azan car-
mine".) Another modification is that of Volkmann and Strauss
(1934) who used azocarmine after a resorcin gentian violet.

There is a closely related dye, azocarmine B (C. I. No. 829)
which is a trisulfonate instead of a disulfonate. Heidenhain em-

Quinone-imine Dyes 133

ployed either, finding a stronger solution necessary in the case of
azocarmine B, which required more application of heat (56°C.) in
order to bring it into solution.

Besides the misspelling "azan carmine" one also frequently sees
the name written as two words "azo carmine." The latter is not
a serious mistake, but it is preferred here to write it as one word,
so as not to imply that it is one of the azo dyes.

For technic of Mallory-Heidenhain "azan" connective tissue stain, see Staining
Procedures, IB3-4.

For technic of MolIIer's quadruple stain, see Id. p. IB3-7.
L35 MAGDALA RED C. I. NO. 857

Synonyms: Naphthalene red. Naphthalene pink. N aphthylamine

pink. Sudan red.

This is a naphtho-safranin, and is a mixture of the monamino
and diamino compounds:

I
/ \

.A

J, I I NH. ^^, H.N

CI

C30H20N3CI; Mol. Wt. 457.941 C30H21N4CI; Mol. Wt. 472.957

{A basic dye; absorption maximum about 52 J^.)

A true magdala red put on the market before 1914, and still sold
by Grubler and Co., under the name of magdala rot echt, is quite
expensive. This same company also sells a product "magdala
rot der Handels," and a similar type has been sold by Coleman and
Bell in this country. This so-called commercial magdala red is an
entirely different dye, erythrosin or phloxine (see p. 195)-an acid
instead of a basic dye, and one of an entirely different group.
Chamberlain (1927) states that his work with magdala red was
done with the latter type, hence actually with phloxine instead
of the dye he thought he was using; his results cannot be dupli-
cated with true magdala red. This means that in the well-known
procedure for staining algae, using this dye in combination with
anilin blue, one should actually employ phloxine, not magdala red.
(See also discussion under phloxine, p. 196.) The same statement
probably applies in the case of Dixon's (1920) magdala red and
light green stain for mycelium in plant tissue.

A magdala red has been employed by Flemming as a nuclear

134 Biological Stains

stain, and by Kultschitzky for staining elastic tissue. No infor-
mation is available, however, as to whether they used phloxine or
true magdala red.

c. The Indulins

Indulins are similar to safranins but are more complex: being
quite highly phenylated amino derivatives. Only a few concern us.

'-40 INDULIN, SPIRIT SOLUBLE C. I. NO. 86o

Synonyms: Indulin (with various shade designations). Spirit

indulin. Spirit nigrosin R.

This consists of mixtures of the following:

HN_/\_N_/\_NH , /\_N_/V_NH

I J I J and •HN_| J f J

HN-\/-N=\/-NH I \/— N=\/-NH

" ^ " 6

C42H33N6CI; Mol. Wt. 657.189 CseHzsNgCl; Mol. Wt. 566.081

{A basic dye; absorption maximum varies from 54-1.5 to 560.5)

l45 indulin, water SOLUBLE * C. I. NO. 86 1

Synonyms: Indulin (with various shade designations). Soluble
indulin SB. Fast blue B, OBy R, 2R, etc.

{An acid dye)

This is the sodium salt of the sulfonation product of indulin,
spirit soluble (C. I. No. 860). Because there are various mixtures
of the latter and different degrees of sulfonation are possible, this
dye may vary greatly in its composition and shade.

One of the indulins (exact nature uncertain) has been used by
Lynch (1930) in the counterstaining of bulk tissue; also by Cum-
ley (1935) in the negative staining of bacteria. For the latter pur-
pose, however, the following dye is more commonly employed.

Quinone-imine Dyes 135

L50 NIGROSIN, WATER SOLUBLE C. I. NO. 865

Synonyms: Nigrosin W, WL, etc. Gray R, B, BB. Silver gray.

Steel gray. Indulin black.

Nigrosin is not a pure dye, but is a mixture; and apparently the
composition of different lots may vary. Ordinarily it is a mixture
of a blue-black or violet indulin with a yellow dye in such a pro-
portion that the resulting blend appears black. As the proportion
of these two dyes is not always the same, different samples of
nigrosin may vary in the amount of blue apparent to the eye.
It is assumed that the biologist wants a nigrosin which appears
distinctly black; this is certainly true in those procedures where it
is used for a background stain. Accordingly the samples of
nigrosin submitted which have a bluish hue have been refused
certification by the Stain Commission.

N'igrosin is used in place of India ink as a background stain in
the study of unstained bacteria ; also as a stain for the background
in contrast to fuchsin in the Dorner (1926) stain for bacterial
spores. It is recommended by Ehrlich for staining the tissue
of the central nervous system either alone or in combination
with other stains, and by Jarotsky for staining pancreatic
tissue following hematoxylin. Botanists use it in studying algae
and fungi. Pfitzer's (1883) picronigrosin serves as a chromatin
stain. Nigrosin is also used by Unna (1921) in combination with
**orange" (orange G.^) in the study of the process of chromolysis.
Bean (1927) has employed it as a counterstain for nervous tissue.

For the use in negative staining of bacteria, see Staining Procedures, p. Ill A2-6,
For technic of Dorner spore stain for bacteria, see Id. p. IIIA2-9.

CHAPTER VII
THE PHENYL METHANE DYES

ONE of the most important groups of dyes, both from the
standpoint of the dyer and from that of the biologist, is a
group of substituted methanes, or in other words compounds
with a central carbon atom. In methane, CH4, it is possible to re-
place any of the hydrogen atoms with methyl, ethyl, or phenyl
groups. If one H is replaced with CH3, it becomes ethane,
CH3CH3. If two are replaced with CH3 groups it becomes pro-
pane, CH3CH2CH3; while if there are three substituent CH3
groups it becomes iso-butane:

CH3 CH3

\ /
C

/ \
CH3 H

Similarly if one H is replaced with a phenyl group it becomes
phenyl methane or toluene:

CH3

if with two it becomes di-phenyl methane:

if with three it becomes tri-phenyl methane:

H

C

Certain substitution products of the di- and tri-phenyl methanes
are among the most powerful dyes known.

Di- and tri-phenyl methane, themselves, are not dyes, nor are
they chromogens. They lack both the chromophore and the auxo-
chrome groups. The first step (theoretically) in converting them
into dyes is to introduce an -OH group in the place of one of the
unsubstituted H atoms of the methane nucleus. The compound
thus formed, which bears the same relation to the phenyl methane
as alcohol does to methane, is called a carbinol. A carbinol is
methyl alcohol in which one or more of the hydrogen atoms may
have been replaced with an alkyl radical or a benzene ring. Thus:

137

138 Biological Stains

It is next theoretically possible to attach amino groups to the ben-
zene rings. Thus in the case of di-phenyl carbinol it is possible to
obtain di-amino di-phenyl carbinol:

H

H2N_/~V_ C_/~\_NH2

OH

Now this latter compound contains the necessary auxochrome
groups; but it is not yet a dye. No carbinol is a dye, because it
lacks a chromophore group. The carbinols are important in dye
chemistry, however, because upon dehydration a rearrangement of
the bonds in the molecule takes place giving the quinoid benzene
ring, which as we have seen is a powerful chromophore. Thus:

H

HaN_/^\_C_/~V_NH, -^ H.N_/— \_C_/~\^NH+H,0

OH H

This latter compound is colored and is the anhydride of a true dye
base. Upon hydration it should theoretically become:

H H

I I

H2N_/~\_C_/—\_N_0H

\_/ \_/ I

H

Such a compound could exist only in aqueous solution. It is
known only by its salts, the true dyes, as:

H

I
H2N _ /-\ C_

Although the theoretical compound given above is the true dye
base, the carbinols are often known as carbinol bases or color bases
of the phenyl methane dyes. As stated above, they lack the
chromophore group, and hence are colorless.

1. DI-PHENYL METHANE DERIVATIVES

The di-phenyl methanes are seldom of biological significance.
Only one deserves mention here.

115

Phenyl Methane Dyes

AURAMINE O

139

C. I. NO. 655

Synonyms: Canary yellow. Pyoktaninum aureum.

Pyoktanin yellow.

CHj

\N_/

CH,

_C_
NH2

./-\_N/

CH:

CH3

CI/
C17H22N3CI; Mol. Wt. 303.827

{A basic dye; absorption maxima [380], J^31-JtSJt)

Solubility at 26°C: in water 0.74%; in alcohol 4.^9%

.8-

.7-

•

.6-

.5-

55

.4-

UJ

.3-

.2-

L 1

330

I I I I I I I I I I I I
360 390 420 450
WAVELENGTH m^

Fig. 17. Spectral curve of auramine O.

I J

480

140 Biological Stains

Auramine O has been in use for some time as a drug, but until
recently has been regarded as having httle value in microscopic
technic. A search of the literature in 1936 revealed only the
following rather obscure staining procedures calling for this dye:
by Vinassa (1891) for staining plant sections; by Fischel (1901)
for vital staining of salamander larvae; and by Kisser (1931), with
cresyl violet as a tissue stain. More recently, however, its pro-
perties as a fluorochrome have brought it into considerable promi-
nence in fluorescence microscopy. Richards and Miller (1941)
recommend it as the best, among numerous fluorochromes investi-
gated by them, for staining the tubercle organism. Their method,
because of the low magnification and large fields which it makes
possible, allows such an increase in the number of positives in
diagnosis that it has attracted much attention to this dye since
their method appeared.

The spectrophotometric curve of a typical sample is given in
Fig. 17. The most striking feature of this curve is the secondary
maximum at 380.

For technic of fluorescent staining of acid-fast bacteria, see Staining Procedures,*
p. IIIA2-9.

2. TRI-PHENYL METHANE DERIVATIVES

There are two groups of tri-phenyl methanes to concern us, the
amino and the hydroxy derivatives. The former, which are much
the more numerous, are very strongly basic, thanks to the amino
groups, unless sulfonated like light green or acid fuchsin. The
rosolic acid dyes, on the other hand, are hydroxy phenyl methanes,
the amino groups being replaced by hydroxyl groups; they are
therefore acid instead of basic dyes.

There are likewise two subdivisions of the amino derivatives,
the di-amino tri-phenyl methanes and the tri-amino tri-phenyl
methanes. These two groups are derivatives respectively of:
di-amino tri-phenyl methane

H2N.

H2N

and tri-amino tri-phenyl methane, or paraleucanilin.

H2N_/-\ n

C

*Conn and Darrow (1943-4).

Phenyl Methane Dyes 141

The individual dyes of this series are substitution products of these
two compounds and differ from one another in the number of
methyl, ethyl, or phenyl groups introduced, and according to
whether they are introduced into the amino groups or directly
onto the benzene rings.

a. Di-AMiNo Tri-phenyl Methanes

m50 malachite green C. I. NO. 657

Synonyms: Victoria green B or WB. New Victoria green extra, 0, /
or II. Diamond green B, BX, or P extra. Solid green 0.

Light green N.

Various brands denoted: Malachite green A, B, BX, 4B, J3E,
J3ES, NB, NH, or NJ.

CH3

/
CH3 CI /~\_N

\ I /\_/ \

N_/~\_C CH3

CH3 \_/

C23H25N2CI; Mol. Wt. 364.903
{Absorption maxima: 617-619, [425])

See Fig. 18 for spectral curve.

Malachite green is a rather w^eakly basic dye that has been used
in the past for various histological purposes; as by v. Beneden for
staining Ascaris eggs, by Petroff for staining erythrocytes, and by
Maas as a contrast stain following borax carmine; see Krause
(1926-7), p. 1353. Today it has very largely been replaced by
methyl green; but it is still called for in special procedures, of
w^hich the following might be listed by way of illustration: w^th
acid fuchsin and martins yellow in the Pianese (1896) technic
which w^as originally applied to cancer tissue but is now much
more widely used in plant pathology; in its reduced (leuco) form,
by Chapman and Lieb (1937), as a reagent (although less sensitive
than crystal violet) for bacterial polysaccharides; as a bacterio-
static or amebicidic agent (Verona, 1935; Tsuchiya, 1936); as a
vital stain for nulei of epidermis stains of the onion (Bank, 1938);
by Schaeffer and Fulton (1933) as a bacterial spore stain; in place
of methyl green in the Pappenheim stain, when combined with the
Gram stain (Sandiford, 1938); also as a constituent of certain
bacterial stains.

For technic of Pianese IIIB stain for fungi In plant tissue, see Staining Pro-
cedures, p. IIA-12.

For technic of SchaeflEer and Fulton method of spore staining, see Id. p. IIIA2-IO.

For technic of Laybourn modification of Albert stain for diphtheria (which calls
for this dye, mixed with toluidine blue), see Id. p. IIIA2-n.

142

Biological Stains

.7-

\

.6-

e

M

e>

5-

o

J

.4-

>-

t

en

3-

"Z,

•*^

Ul

o

.2-

oc

o

-J

l->^

o

X ^

o

^ >

L • 1

400

I I I I I I I I J
460 520 580 640 700
WAVELENGTH m )i

Fig. 18. Spectral curves of two phenyl methane dyes:
1. Brilliant green. 2. Malachite green.

M.55

BRILLIANT GREEN

C. L NO. 662.

Synonyms: Ethyl green. Malachite green G, Emerald green
crystals.* Solid green J JO. Diamond green G.

This is a basic dye which is generally known in the form of the
acid sulfate:

CH2CH3

CH3CH. SO,H

\l

N

_ /~\=C CH2 CH3

CH3CH. \_/

C27H34N2O4S; Mol. Wt. 482.618
(Absorption maximum: 628-631, [^25])

"Malachite green, also, has been exported from Germany under this name

Phenyl Methane Dyes 143

Brilliant green is used to some extent as a stain for bacteria,
spirochaetes, molds, and yeasts (see, for instance, Krajian, 1941),
but finds more frequent employment as a constituent of bacterio-
logical media. It is used, for example, as follows; (1) as a con-
stituent of Krumwiede's brilliant green bile media for plating
water in order to distinguish the colon organism from other lactose
fermenting organisms; (2) it is employed in media used in search-
ing for the typhoid organism in stools, where its value comes from
its ability to inhibit the colon organism; (3) it is used as a con-
stituent of enrichment media for the stimulation of the typhoid
organism, in which it must prevent the growth of the normal colon
types but not be toxic to the typhoid organism. It has proved
much more simple to find a brilliant green satisfactory for colon
diagnosis in water work than for the isolation and enrichment of
the typhoid organism. A paper by Rakieten and Rettger (1927)
has shown the difficulty encountered in this latter instance.

As the first of three purposes above mentioned is the one for
which brilliant green is now most commonly used in America, all
samples submitted for certification are tested for use in the bril-
liant green bile medium.

Spectrophotometric curves of typical samples of malachite
green and brilliant green are given in Fig. 18. All green dyes have
two absorption maxima, one about 430, the other about 620-630.
The primary maximum for malachite green, however,, is at a defi-
nitely shorter wavelength than that of brilliant green or either of
the two following dyes.

m60 light green SF YELLOWISH C. I. NO. 67O

Synonyms : -Light green 2G, S or 2GN. Acid green (with various
shade designations). Fast acid green N.

This is a derivative of brilliant green, which is sulfonated and is
therefore an acid dye.

NaS03_

C37H34N209S3Na2 ^Afoi. Wt. 792.832

(Absorption maximum: 629-634, [428])

SdvinlUy at 26^0: in water 20.35%; in alcohol 0.82%

Light green is a valuable plasma stain often used for staining
animal tissues in contrast to iron hematoxylin or other nuclear
dyes, although it fades badly if exposed to bright light. It is a

144

Biological Stains

very valuable counterstain to safranin, especially after Flemming
fixation, thus finding employment in cytological work. It
photographs well. It is a constituent, together with neutral red,
of the compound dye employed in the Twort (1924) stain for
microorganisms in tissues and has been applied to the staining of
bacteria, yeasts, algae, etc., under various conditions. In plant
histology it is a useful cytoplasm and cellulose stain and has been
employed by Buchholz (1931), mixed with acid fuchsin, for staining
pollen tubes. Its greatest drawback is that it fades rapidly and is
therefore not very permanent. Where greater permanency is de-
sired the following dye may often be substituted for it.

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH THIS STAIN IS USED*

NAME OF PROCEDURE

page reference to
Staining Procedures

Grossman's modification of Mallory collagen stain
Counterstain to Heidenhain's hematoxylin for plant tissue
Safranin and light green in plant histology
Counterstain to Feulgen stain for nucleoli In plant cytology

IB3-5
IIA-7
IIA-10
IIB-14

*Under this heading are given references to procedures described in detail in
Staining Procedures, edited by Conn and Darrow (1943-4).

m65

FAST GREEN FCF

This is a dye, very closely related to light green SF yellowish,
which was originally proposed by Johnson and Staub (1927) as
a food dye.

NaSO,

CH3 CH,

CH,

.N_/-

CH2'CHj

/

/ I \CH._/— \_SO3Na
SO3 \_/

OH

C37H34N20ioS3Na2; Mol. Wt. 808.832

{An acid dye; absorption maxima about 625 , [420-430])

Solubility at 26° C: in water 16.04%; in alcohol 0.35%

This dye was first tried in the Commission laboratory as a sub-
stitute for light green SF yellowish (see Haynes 1928) and has
now come into general use in plant histology and cytology. It
gives staining effects very much like light green and is consider-

Phenyl Methane Dyes

145

ably less subject to fading. Slides have been exposed to direct
sunlight for a few weeks and have still retained the green color.
This dye is therefore to be recommended for such use as it has all
the advantages of light green without the disadvantages. Con-
sidering the comparatively short period of time since this dye was
first introduced to the microscopist, it is now employed in a sur-
prisingly large number of procedures, among which might be men-
tioned the following as illustrating two widely different types of

.7-

N.6-

o
H

O

.5-

£.3-

UJ

o

O

o
o

380

I I I I I I

440 500 560 620
WAVELENGTH TTI^A

I J

680

Fig. 19. Spectral curves of three phenyl methane dyes:
1. Methyl green. 2. Light green SFY. 3. Fast green FCF.

methods: the quadruple staining technic of Johansen (1939), for
plant histology, in which it is combined with safranin, methyl
violet, and orange G; its use as a bacterial stain by Maneval,
(1941), who recommends this and other acid dyes (e.g. acid fuch-
sin, aniline blue W.S., and orseilline) as preferable to the conven-
tional basic dyes because of the better diflFerentiation afforded and
the less tendency for slime and debris to take the stain.

146 Biological Stains

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH THIS STAIN IS USED

NAME OF PROCEDURE

page reference to
Staining Procedures

Kornhauser's "Quad" stain for tissue

Modification of Van Gieson stain

Lillie modification of Masson trichrome stain

Safranin and fast green in plant cytology

De Tomasi modification of Feulgen stain in plant cytology

IB3-8
IB3-II
IB3-I9
IIB-11
IIB-13

Spectrophotometric curves of typical samples of light green
and fast green are given in Fig. 19. It will be seen that they are
both very much like the curve for brilliant green, although slightly
different from malachite green in the location of the primary
absorption maximum.

Another dye of this group which has been mentioned in biolog-
ical literature is:
Acid green L Extra; C. I. No. 666. Syn. : Acid green G, B extra,

GV. Pontacyl green B.

b. Tri-amino Tri-phenyl Methanes (Rosanilins)

The simplest rosanilins are the dyes sold as basic fuchsin. This
term is somewhat loosely used to apply to two or three different
dyes or to various mixtures of them. The dyes known as fuchsin
differ from the methyl violets and other rosanilins in that the
amino groups are not methylated or substituted in any other way.
The fuchsins may, however, have methyl groups introduced
directly onto the benzene rings instead of into the amino groups ; and
the different fuchsins vary from one another in the number of
such methyl groi^jps present. There are four primary compounds
theoretically possible, namely with no methyl group, and with
one, two, and three substituent methyl groups respectively.

BASIC FUCHSIN

Synonyms: Fuchsin RFN. Magenta. Basic ruhin. Anilin red*

The dyes ordinarily known to commerce as basic fuchsin are
represented by C. I. No. 677, which is a mixture of pararosanilin,
rosanilin and magenta II. Pararosanilin is obtainable in fairly
pure form, as is also the compound with three substituent methyl
groups, namely new fuchsin. The formulae of these compounds
follow :

*An impure basic fuchsin known as cerise has been mentioned in the biological
literature.

Phenyl Methane Dyes 147

n2 Pararosanilin (Magenta O) c. i. no. 676

Synonyms: Basic rubin. Parafuchsin. Paramagenta.

This compound is triamino-triphenyl-methane chloride.

H CI /"~\_NH.

\ I ,_, /\_/
N=/^\=C _

/ \_/ \/ \_NH3
H \_/

CgHisNsCl; MoL Wt. 323.815

{A basic dye; absorption maximum about 5^)

Solubility at 26°C: in water 0.26%; in alcohol 5.93%

It is the chief constituent of the majority of samples of basic
fuchsin submitted for certification as biological stains. It is
sometimes furnished as the chloride, sometimes as the acetate,
generally the latter.

n3 Rosanilin (Magenta I)

This compound is mono-methyl fuchsin, or triamino-tolyl-
diphenyl-methane chloride.

CH3

H CI

/ \_/

H

C20H20N3CI; Mol. Wt. 337.841

{A basic dye; absorption maximum about 550)
Solubility at 26°: in water 0.39%ci; in alcohol 8.16%

It is not a textile dye, and is not found free from pararosanilin
unless specially prepared.

n4 Magenta II

This theoretical constituent of basic fuchsin is never encountered
pure except as specially prepared in the laboratory, but probably
is present in market samples of the dye. It is di-methyl fuchsin,
or triamino-ditolyl-phenyl-methane chloride.

148 Biological Stains

CTI3

C21H22N3CI; Mol. Wt. 351.867

{A basic dye; absorption maximum about 55 J/)

n5 New Fuchsin (Magenta III) c. i. no. 678

Synonyms: Isorubin. Fuchsin NB.

This compound is tri-methyl fuchsin, or triamino-tritolyl-
methane chloride:

CH3

I
H CI /""\ NH.

N_/~\_C

_. „ ~\_NH2

H I \^/

CH3 I

CHa

C22H24N3CI; Mol. Wt. 365.893

{A basic dye; absorption maximum about 556)

Solubility at 26° C: in water 1.13%; in alcohol 3.20%

From the general rule concerning alkyl substitution it will be
understood that new fuchsin is the deepest in shade of these four
dyes, pararosanilin the least so. The commercial fuchsins, there-
fore, which consist of mixtures of the first three vary in depth ac-
cording to the proportions in which they are present.

A typical spectrophotometric curve of rosanilin (Magenta I)
is given in Fig. 20. Those for Magentas O, II, and III are not
given here, as they are so nearly the same that they can be dis-
tinguished only by careful measurement, and the differences
would not show in such a graph as those here included.

It has proved from the investigations of the Stain Commission
that not all basic fuchsins furnished as biological stains are mix-
tures of these three compounds. Many of the samples prove to be
fairly pure pararosanilin; while others are deeper in shade than
rosanilin and presumably contain appreciable quantities of
magenta II. All of these dyes differ so very slightly from each

Phenyl Methane Dyes

149

other in hue that it takes very careful comparison to distinguish
one from another, and for many purposes they may be used inter-
changeably.

The basic fuchsins are among the most powerful nuclear dyes,
and find many biological uses. They are valuable stains for mucin,
for elastic tissue, and for bringing out the so-called fuchsinophile

.8-

o
o

L I I I I I I I I I I I I I I J
430 460 490 520 550 580

WAVELENGTH m>l

Fig. 20. Spectral curves of two rosanilins:
1. Basic fuchsin. 2. Acid fuchsin.

granules. A basic fuchsin is often employed for staining the nu-
clear elements of the central nervous tissue. This group of dyes is
almost indispensable in bacteriology, particularly in the Ziehl-
Neelsen method, with its various modifications, for differentiating
the tubercle organism and thus making possible the diagnosis of
tuberculosis.

150 Biological Stains

Basic fuchsin is also employed as a chemical reagent in a deter-
mination which is coming to be of considerable interest to the
biologist. Basic fuchsin is the main constituent of Schiff's re-
agent, often employed for the detection of aldehydes, although not
an absolutely specific reagent for the purpose. In preparing this re-
agent, basic fuchsin is reduced through the action of sulfite to a
colorless form, having the type of the formula for leuco-fuchsin
which is given at the middle of page 31, (The formula there given
for leuco-fuchsin, however, is not exactly that of Schiff's reagent, as
it is believed that the sulfite radical in some way enters into its com-
position.) In the presence of aldehyde some chemical reaction not
wholly understood takes place which evidently restores the quino-
noid structure of the molecule and, accordingly, the color of the
compound. Apparently a slightly different dye from the basic
fuchsin is thus produced, since the color is violet rather than pure
red.

This reaction is of interest to the bacteriologist, as basic fuchsin
is employed similarly in the Endo medium for distinguishing be-
tween members of the colon-typhoid group of bacteria. This
medium contains lactose, which is not acted upon by the typhoid
organism but is fermented by organisms like Bacterium call. It
has been known for many years that these lactose fermenting
organisms of the group restore the red color of the medium, while
the non-lactose-fermenters do not. The suggestion was naturally
made that this Endo reaction might depend upon the production
of aldehyde by the organisms, but for years no one was able to
demonstrate the presence of aldehyde in such cultures. Neuberg
and Nord (1919) suggested, however, that aldehyde might be
produced but not ordinarily accumulate in sufficient quantity to
be detected; and that the sulfite in the medium might act as a
''trapping agent'* so as to allow it to accumulate. This has since
been verified by Margolena and Hansen (1933). Apparently,
therefore, the Endo medium is a biological application of Schiff's
reagent.

Feulgen and his associates (Feulgen and Rossenbeck, 1924;
Feulgen and Voit, 1924) employed this compound of basic fuchsin
and sulfurous acid in histological technic, regarding it as a micro-
chemical reagent for detecting the presence of aldehyde-like sub-
stances in the nuclei. Feulgen called this the "nucleal reaction,"
the ending "-al" indicating the aldehyde-like nature of the sub-
stance which he considered the reaction to demonstrate. This
interpretation of the Feulgen reaction is now regarded as only
part of the story; the significance of the desoxyribosenucleic acid
demonstrated by the method is appreciated more today than it
was when Feulgen proposed the method.

As a matter of fact, the Schiff reagent, employed by some modi-
fication of the Feulgen technic, has given histochemistry its

Phenyl Methane Dye^ 151

greatest impetus. Special significance has attached to the HIO4-
Schiff technic, in which the use of the Schiff reagent is preceded by
oxidation with periodic acid (HIO4), as first proposed by McManus
(1946). It is considered that certain polysaccharides (and their
derivatives) which contain 1,2-glycol and a-amino-alcohol groups
react with the periodic acid (or other oxidant) to produce aldehyde
groups, and that the latter react with the Schiff reagent to bring
out the violet color. This has enabled histochemists to deter-
mine the locations of the compounds in question within the cell.
Histochemists are proving much more exacting in their require-
ments for a basic fuchsin than were histologists or chemists using
the Schiff reagent. As a result of trying to meet their require-
ments, the quality of basic fuchsin on the American market has
been improving in recent years.

Early editions of this book contained statements, realized at the
time to be largely provisional, to indicate which types of fuchsin
are most satisfactory for the various purposes. Data that have
been accumulated since 1929, however, seem to show pretty
definitely that the exact chemical nature of basic fuchsin itself is
of comparatively little importance. Good results for practically
any purpose have been obtained with pararosanilin, rosanilin,
new fuchsin, and even with the seldom-encountered magenta II;
and the dyes have proved equally satisfactory whether in the
form of chlorides or acetates, the two most common salts of this
dye. From the standpoint of ordinary staining, in fact, it was
realized a long while ago that the samples furnished by the stain
companies varied very little in their performance.

In their behavior in the Endo medium and in the Feulgen
technic, however, the samples were found to be quite dissimilar,
and it was some time before any clue could be obtained as to the
cause of their differences. It has finally been concluded, however,
that the variations which occur are due to foreign ingredients
which are almost always present in these dyes.

The problem of the Endo medium was solved first. It was
learned (Conn and Darrow, 1934) that almost any sample of
basic fuchsin will give good results in the Endo medium if the
formula of that medium is carefully adjusted so as to avoid either
an excess or a deficiency of sulfite in proportion to the fuchsin.
Another complication arises from the fact that the 1936 edition of
the Standard Methods of Water Analysis (of the American Public
Health Association) lists two different formulae for the Endo
medium, of which Formula II calls for K2HPO4, while formula I
does not. For some reason, not yet explained, it is possible for a
particular sample of basic fuchsin to give good results in Formula
II while appearing too deeply colored in Formula I; or to be satis-
factory in Formula I but not allowing sufficient color restoration
by lactose fermenters if Formula II be used. As a result it is

152

Biological Stains

necessary to select between these two formulae the one best suited
to the particular batch of fuchsin to be employed. It is also well
recognized today that many basic fuchsins contain an impurity
which does not completely decolorize under the action of sulfite,
and which becomes evident in ^uch a reduced solution by the
yellowish, or even brownish pigment remaining. The nature of
this pigment has not been learned, altho Scanlan and Melin (1937)
report an investigation of the subject. Following the publication
of Scanlan and Melin's paper, the stain companies have begun to
find ways of preparing fuchsins that are free from the undesirable
impurity; and batches submitted for certification since then have
ordinarily proved satisfactory for the Feulgen technic as well as
for the other purposes discussed above. Not long after the above
paper appeared Coleman (1938) reported a method of eliminating
this impurity from a decolorized fuchsin by agitation with a de-
colorizing carbon; his method seems to make a sample of this dye
usable in the Feulgen technic, even though previously entirely un-
satisfactory. The histochemists, however, want a glass-clear
Schiff reagent; and modern producers of stains in America are
doing their best to supply basic fuchsins that will suit. This is at
present one of the tests applied to samples submitted for certifi-
cation. Lillie (1951) has contributed considerable information
on the subject.

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH THIS DYE IS USED

NAME OF PROCEDURE

PAGE REFERENCE TO

Staining Procedures

HIO4 leucofuchsin method for glycogen

IA-15

Weigert's resorcin fuchsin

IB3-I3

Lillie modification of Gallego elastic tissue stain

IB3-I5

Bauer's leucofuchsin method for glycogen

ID3-22

Aceto-carmine and basic fuchsin for pollen tubes in style

IIA-13

DeTomasi modification of Feulgen stain in plant cytology

IIB-13

Feulgen stain for nucleoli in plant cytology

IIB-14

General staining of pure cultures of bacteria

IIIAo-5

Ziehl-Neelsen stain for acid fast bacteria

IIIA2-8

Domer's spore stain for bacteria

IIIA2-9

Snyder modification

IIIA,-10

Casares-GiFs method for bacterial flagella

inA2-14

Gray's method for bacterial flagella

IIIA2-I5

Liefson's method for bacterial flagella

lUAo-\5

Bailey's method for bacterial flagella

IIIA.-16

Hiss' method for bacterial capsules

IIIA2-I7

Macchiavello's stain for Rickettsiae

IIIA2-I9

Weigert's stain for fibrin and bacteria in tissues

IIIB3-9

VerhoeflF's carbol fuchsin for tubercle bacilli In tissues

IIIB3-IO

Lillie modification of Ziehl-Neelsen technic

IIIB3-II

MacCallum's stain for Influenza bacilli in tissues

IIIB3-I4

Williams' stain for Negri bodies

IIIB3-I3

Goodpasture's stain for influenza bacilli In tissues

IIIB3-I5

Phenyl Methane Dyes 153

One comparatively recent complaint concerning certain samples
of basic fuchsin relates to their behavior in staining tubercle bac-
teria by the acid-fast technic. It has been claimed that with cer-
tain samples of the dye these organisms show a tendency to de-
colorize partially or wholly, thus resulting in poorly stained cells
that tend to appear as a string of beads. This has proved a
difficult complaint to investigate, because consistent results are
not obtained by different investigators. In fact, Yegian and
Porter (1944) have shown that a normally staining culture may be
converted into one which is not acid-fast or which shows beaded
staining by improper methods of smearing on the slide. This
observation makes it appear doubtful whether the complaints in
question may or may not be due to variations between the different
lots of fuchsin.

In general it can be stated, that in spite of all variations re-
ported, the standardization of this dye seems to have been accom-
plished in a practical way, even if there is still much to be learned
about it from a theoretical standpoint.

Nl2 ACID FUCHSIN C. I. NO. 692.

Synonyms: Fuchsin S, SN^ SS, ST y or S III. Acid magenta.

Acid ruhin.

{An acid dye; absorption maximum: pararosanilin 5J!^0~5J^3,

rosanilin 5J^3-5Jf6)

This dye owes its acid character to the fact that it is a sulfonated
derivative of basic fuchsin. Acid fuchsins are ordinarily rather
complex mixtures. As there are four primary basic fuchsins pos-
sible, according to the degree of methyl substitution, and as each
may yield at least three different compounds on sulfonation, fully
a dozen acid fuchsins are theoretically possible, and samples are
hardly to be expected which are not mixtures of several.

The generally accepted formula of one of the homologs present
in acid fuchsin, namely the di-sodium salt of rosanilin trisulphonic
acid, is:

SOs-Na

/
CH3 /-\_NH2

H2N_/~\_C SO3

\_/ /

/ /-\_NH2

NaSOs \_/

C2oHi7N309S3Na2; Mol. Wt. 585.534

The bond connecting one of the sulfonic groups with an amino
group attached to a different benzene ring is assumed to exist in

154 Biological Stains

order to account for the fact that although only two of the sulfonic
groups are neutralized with sodium, the compound acts as though
it has no free acid. In other words, it is a case of intramolecular
salt formation. Now w^hen the tri-sodium salt is formed, this bond
is broken down, whereupon the quinoid ring disappears and the fol-
lowing compound is produced :

SOsNa

/

/~\_NH2

\ /

H,N_/— \_C— OH SOaNa

\_/ \ /

/ \/~\_NH2

NaSO

3

This compound, it will be seen, is a carbinol in structure, and as it
lacks the quinoid ring it is colorless; but it is very readily converted
into the di-sodium salt by the addition of acid, whereupon the color
again appears. This property makes acid fuchsin of use as an
indicator. The decolorized solution of acid fuchsin neutralized
with sodium hydroxide is called the Andrade indicator. It was
once used extensively in bacteriological work, because of the strik-
ing reaction when its color is restored by acid-forming bacteria.
(See Andrade-Penny, 1895.) As an indicator to show hydrogen-
ion concentration at all accurately, however, it is found to have
much less value than the phthalein and sulphonphthalein dyes (see
pp. 197 to 206).

Acid fuchsin is a widely used plasma stain, which has also been
recommended for a number of special uses. Among the best
known are: the Van Gieson (1889) connective tissue stain, in which
it is used with picric acid after hematoxylin to differentiate smooth
muscle from connective tissue; the Ehrlich-Biondi tri-acid stain
for blood, which is a "neutral" combination with orange G and
methyl green (see Krause, 1926-7; pp. 457, 1707). In plant his-
tology it is used to stain the cortex, pith and cellulose walls:
while the Pianese (1896) stain (with malachite green and martins
yellow), which was originally applied to cancer tissue, is now used
by plant pathologists in studying infected vascular plants. It is
used with methyl green, by Altmann, Bensley and Cowdry as a
stain for mitochondria (see Lee, 1937, pp. 167-8). To the pathol-
ogist it is quite valuable as a constituent (with anilin blue and
orange G) of the Mallory (1900) connective tissue stain.

Scanlan, French and Holmes, (1927) showed that many lots of
acid fuchsin are unsatisfactory because of the rapidity with which
they fade in Van Gieson preparations. These writers found that
this fault arises from lack of control of the process of sulfonation.
Their results indicated that a satisfactory product could be ob-
tained if this process were carefully controlled by the use of a low

Phenyl Methane Dyes 155

temperature (85°C.), fuming sulfuric acid containing sufficient
free SO3 to insure complete sulfonation to the trisulfonic acid, and
careful acidification to yield only the disodium salt. Since the
method which they proposed in this article was called to the atten-
tion of manufacturers, the American product has improved; it not
only gives permanent Van Gieson preparations, but proves more
satisfactory in the Mallory connective tissue stain.

The spectrophotometric curve of a typical acid magenta I
(rosanilin) is given in Fig. 20 (curve 2), p. 149. The chief dif-
ference between this and the corresponding basic fuchsin is that it
has a distinct secondary maximum at about 500, where the curve
for basic rosanilin shows merely an almost imperceptible bulge.
A dye having its primary maximum at 500 would be orange in
shade, which is approximately the color of the acid phase of the
Andrade indicator.

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH THIS STAIN IS USED

PAGE REFERENCE TO

NAME OF PROCEDURE

Staining Procedures

Van Gieson stain with iron hematoxylin

IA4-21

Mallory's anilin blue collagen stain

IB3-3

Grossman's modification

IB3-5

Acid alizarin blue modification

IB3-6

Masson's trichrome stain

IB3-I8

Pianese IIIB stain for fungi in plant tissue

IIA-12

n20 HOFMANN S violet C. I. NO. 679

Synonyms : Dahlia. Iodine violet. Red violet. Violet R, RR or

4RN. Primula R water soluble.

These various names are applied in a somewhat indiscriminate
way to dyes intermediate in shade between basic fuchsin and
methyl violet. Theoretically they are mixtures of methylated
and ethylated pararosanilins and rosanilins, having fewer than
five ethyl or methyl groups. Of these compounds, the formula
for tri-ethyl rosanilin is:

CI

I

1 ^
H

H

C26H32N3CI; Mol. Wt. 421.997
{A basic dye )

156 Biological Stains

In actual practice, however, the names applied to this group
of dyes are very loosely employed, and one is often furnished
under such a label as dahlia or Hofmann's violet a mixture of basic
fuchsin and methyl violet, having a shade about the same as one
of the compounds just mentioned. Two samples of * 'dahlia"
have been called to the attention of the Commission which actu-
ally differed very greatly, one being nearly as red as fuchsin, the
other nearly as blue as methyl violet. There was every reason to
believe that one was a fuchsin with a small addition of methyl
violet, the other a methyl violet with a little fuchsin added. The
latter dyes are much more cheaply prepared than the true Hof-
mann violets, and the substitution is natural considering that the
shade obtained can be the same.

Hofmann's violet has been called for by Ehrlich and by Unna
for staining mast cells; by Juergens for staining amyloid, which it
colors red, while the cytoplasm is colored blue. (See Krause,
1926-7, p. 1123). Now it is possible that for one of these pro-
cedures or for some other similar one, a true Hofmann's violet is
necessary; on the other hand the shade obtained may be the im-
portant matter, in which case as satisfactory results should be ob-
tainable with a mixture of fuchsin and methyl violet. This is a
matter that should be further investigated; for if the cheaper
fuchsins and methyl violets can be substituted for the Hofmann
violets, the substitution can be made by the biologist himself. It
is interesting to remark that one supply house not long ago sent out
a bottle of crystal violet, unintentionally mislabeled dahlia, to a
biologist ordering the latter dye. This biologist shortly wrote to
the company that he would like more of it, as it was the best lot of
dahlia he had ever used; in looking the matter up to duplicate the
former order, the company discovered its mistake.

It is extremely difficult to determine just what dye in this group
is referred to by any given name. Thus a rosanilin violet has
been mentioned as having been employed in a stain for Negri
bodies; and it is uncertain whether it is a dye of the above group or
of the group following. Any biologist employing one of the violet
rosanilins should be especially careful in publishing his work to
give all the information furnished on the label as to manufacturer
and the nature of the dye; and should preferably verify all such
work, before publication, by using a dye of known composition.

n25 methyl violet c. I. NO. 68o

Synonyms : Dahlia B. Paris violet. Pyoktaninum coeruleum.

Gentian piolet.

Various shades denoted: Methyl violet 2R, R, B, 2B, 3B,
BBN, BO, V3.

Phenyl Methane Dyes

157

The various dyes denoted methyl violet are mixtures of tetra-,
penta-, and hexa-methyl pararosanilin :

CH3

CH,

tetra-methyl pararosanilin
C23H26N3CI; Mol. Wt. 379.919

CH, CI

CH,

penta-methyl pararosanilin
C24H28N3CI; Mol Wt. 393.945

CH, CI

hexa-methyl pararosanilin
(crystal violet)

C25H30N3CI; Mol. Wt. 407.971

{Basic dyes; absorption maxima: 583-587 in 90% alcohol)

Solubility at 26°C: in water 2.93%; in alcohol 15.21%o

In the case of these compounds, as in the case of other series of
homologs differing in extent of methylation, the shade is deepened
by the introduction of each methyl group. Hence the various
mixtures known to the trade as methyl violet vary from reddish to
bluish violets according to the relative amounts of the more and
the less completely methylated compounds present in the mixture.
This is the significance of the various shade designations listed
above, R's indicating the reddish shades, and B's the bluish shades.
Of these various shades the bluer ones seem to be best for biological

158 Biological Stains

purposes, methyl violet 2B having been found satisfactory for
practically all purposes for which methyl or gentian violet is
ordinarily called for. This indicates that the biologist requires the
higher homologs in this group. Now the most completely methy-
lated methyl violet is the hexa-methyl compound, which is easily
obtained pure and is known to the trade as crystal violet. This
dye, in fact, is coming to be the one member of this group in most
common use in biology.

Spectrophotometric curves of typical samples of crystal violet
and methyl violet 2B are given in Fig. 21. Their great similarity

o 400 460 520

WAVELENGTH Tn>l

Fig. 21. Spectral curves of two violet rosanilins:
1. Crystal violet. 2. Methyl violet 2B.

is apparent, but the two products can be distinguished by accurate
measurement of the absorption maximum.

Gentian violet. A poorly defined mixture of violet rosanilins is
well known to biologists under the name gentian violet. The name
is not used at present in the dye or textile industries. It apparently
applied originally to a certain mixture containing about half
dextrin and half dye, the dye being a methyl violet, that is a
mixture. of crystal violet with lower homologs of the same series.
The statement was formerly common in biological literature that
gentian violet is a mixture of crystal and methyl violet; but the
looseness of the statement is evident when it is realized that crystal

Phenyl Methane Dyes 159

violet is a component of all the deeper shades of methyl violet.
It is possible that before 1914 gentian violet did represent a fairly
constant mixture, but there seems to be some doubt even on this
point. It is certain that immediately following the first World
War each company used its own judgment as to what to furnish
when gentian violet was ordered. As a result there were placed
on the market under this name various methyl violets, with or
without dextrin, and also crystal violet; of course the purchaser had
no knowledge as to what he was obtaining in any given instance.

Under the circumstances the Commission faced a difficult
problem in trying to standardize gentian violet. The question
was whether to recognize the name at all, or to approve some
particular dye or mixture of dyes of this group as gentian violet.
The former course was almost impossible because of the wide
demand among biologists for a stain labeled gentian violet; it was
plain that the stain companies would meet this demand in some
way. The second course (unless considerable latitude were recog-
nized) would be entirely arbitrary, inasmuch as no information
was available to show which members of this group of dyes were
especially needed in histology or bacteriology. Accordingly in
the first edition of this book gentian violet was defined as either
penta-methyl or hexa-methyl pararosanilin, or else a mixture of
methylated pararosanilins composed primarily of the two com-
pounds just named and having a shade at least as deep as that
recognized in the trade as methyl violet 2B.

This definition was quite broad and did not exclude anything
sold at that time as gentian violet. As a result there are still
various different products sold under this name. The nature of the
imported gentian violet has not been recently investigated ; but it is
known that there are at least three types of gentian violet on the
market, prepared by American concerns. They are as follows:
1) crystal violet, as sold under the labels of the National Aniline
and Chemical Co. and of the Hartman-Leddon Co., each of these
products being plainly marked '^crystal violet" in small letters
following the name "gentian violet;" 2) one of the higher methyl
violets prepared by Coleman and Bell and labeled "gentian violet
improved;" 3) a product manufactured by the National Aniline
and Chemical Co. and labeled "gentian violet. Churchman,"
which is claimed to be a mixture in equal parts of crystal violet
and methyl violet 2B.

The situation is not yet regarded as entirely satisfactory. Users
should specify crystal violet, for bacteriological work and for*
histological purposes where a deep blue-violet is required; but
should order methyl violet 2B in histological procedures where a
reddish shade is called for. With this knowledge at hand, gentian
violet, as such, is not necessary.

160 Biological Stains

N30 CRYSTAL VIOLET C. I. NO. 68 1

Synonyms: Violet C, G, or 7B. Hexamethyl violet. Methyl violet

lOB. Gentian violet.

C25H30N3CI; Mol. Wt. 407.971

{A basic dye: absorption maximum 589-593)

Solubility at 26°C: in water 1.68%; in alcohol 13.87%

This dye is a hexa-methyl-pararosanilin, whose formula is given
above as one of the components of methyl violet. The Commis-
sion has made as careful an investigation of this dye as of any other
and recommends it highly.

Methyl or gentian violet is of chief value to the biologist as a
nuclear or chromatin stain, having many histological and cyto-
logical applications, the one for which it is most commonly used at
present being the Flemming (1891) triple stain in which it is em-
ployed with orange G and safranin — a technic which gives a very
high degree of cytological differentiation. It is also used for
staining amyloid in frozen sections of fresh and fixed tissue, and
for staining the platelets in blood while it is much used by the
Weigert (1887) technic for staining fibrin and neuroglia. The
bacteriologist also finds it a useful stain and probably purchases
more at the present time than all other biologists together; the
chief bacteriological use is in the Gram technic for distinguishing
between different kinds of bacteria. A further more recent use is
in bacteriological media for inhibiting the growth of Gram-positive
organisms, due to its selective bacteriostatic action. An important
example is Petroff's (1915) gentian violet egg medium for isolation
and cultivation of the tubercle organism. For most of these pur-
poses, crystal violet is in general use. As a result of its bacterio-
static properties it has come into use as a medicinal agent for the
control of staphylococcic infection. It has not yet been definitely
established, however, whether crystal violet a.lone or some mixture
thereof with the lower homologs should be used for this purpose.

The Flemming and Gram stains have seemed the most delicate
procedures for which it is used; so they have been given the most
careful study. In the case of the Gram stain it was discovered that
there are a score or more different procedures all referred to by the
name "Gram** stain, and a study was made of all the methods that
were found (see Hucker and Conn, 1923). The result of the in-
vestigation was to conclude without reservation that crystal violet
may be substituted for gentian violet in both the Gram and Flem-
ming technics, and probably for gentian or methyl violet in any of
the bacteriological or histological methods for which either stain
is designated. The advantage of crystal violet is obvious; for it
is a definite chemical compound, while methyl and gentian violet
are both variable mixtures.

It is of interest to note that in the literature of microscopic tech-

Phenyl Methane Dyes

161

nic crystal violet has been specified instead of gentian violet for
some special procedures. Worth noting is Benda's crystal-violet-
alizarin method for staining chondriosomes, and its modifications
by Meves and Duesberg (1908); and also its use in combination
with erythrosin by Jackson (1926) for staining lightly lignified
walls, in which technic it proves more uniform than gentian violet.
A loose compound of crystal violet and bismuth, formed by
treating an aqueous solution of the dye with an aqueous solution
of bismuth ammonium citrate, has been prepared by Wilkinson
and Barksdale (1928) and called bismuth violet. It is claimed to
have much greater bacteriostatic action on Gram-positive bacteria
than crystal violet itself.

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH THIS STAIN IS USED

NAME OF PROCEDURE

PAGE REFERENCE TO

Staining Procedures

Crystal violet stain for amyloid

IA4-9

Jackson's crystal violet stain for woody tissue

IIA-9

Newton's crystal violet iodine technic for plant cytology

IIB-5

Randolph's crystal violet iodine technic for plant cytology

IIB-5

Permanent smear preparations of sporocytes

IIB-6

Flemming triple stain in plant cytology

IIB-10

Same, with iodine

IIB-11

General staining of pure cultures of bacteria

IIIAo-5

Gram stain: Hucker modification

IIIA2-6

Burke and Kopeloff-Beerman modifications

IIIA2-7

Ljubinski stain for diphtheria organism

IIIA.-12

Anthony's method for bacterial capsules

IIIA2-I7

Hiss' method for bacterial capsules

IIIA2-I7

Tyler's method for bacterial capsules

IIIA2-I8

Gram-Welgert method for bacteria in tissues

IIIB3-8

Lillie modification of Gram stain for tissues

IIIB3-8

Weigert's stain for fibrin and bacteria in tissues

IIIB3-9

MacCallum's stain for influenza bacilli in tissues

IIIB3-U

n35 ethyl violet C. I. NO. 68l

Synonym: Ethyl 'purple 6B.

Ethyl violet is hexa-ethyl pararosanilin having the following
formula:

C2H5

CI

I
C,H5-N_/

I N
C2H5

N

N

C2H5
C2H5

C0H5

C31H42N3CI; Mol. Wt. 492.127
{A basic dye; absorption maximum about 596)

162 Biological Stains

It has been called for by Bowie (1924) in a neutral stain com-
bination with Biebrich scarlet to stain the islets of Langerhans.
Kernohan (1931) and Proescher (1934) have each employed it in
staining nervous tissue; while Ono (1934) uses it for staining
spirochaetes in blood.

n40 methyl green C. I. NO. 684

Synonyms: Double green SF. Light green.

Methyl green is crystal violet into which a seventh methyl group
has been introduced by the action of methyl chloride or methyl
iodide upon it, forming the compound:*

CI CH3

/

/

N— CH3

/\-

_/

\

/

CH3

c

CH3

\ _

/

\/

"v

N

_/

CH3

CH3 CI

\l

N.
/

CHa

C26H33N3CI2; MoL Wt. 458.462
{A basic dye; absorption maximum 630-63 J^^ [J^20])

As the seventh methyl group is very loosely attached, there is
always some methyl or crystal violet present, either because it is
not all converted into the higher homolog or because it has broken
down again. It has been stated that to obtain free methyl green
the commercial dye should be shaken in a separatory funnel with
amyl alcohol or chloroform, which dissolves the methyl violet. As
a matter of fact, however, pure methyl green may not always be
desired by the biologist, as the dye owes part of the metachromatic
properties for which it is prized to the presence of small amounts of
the violet compound.

Methyl green is at present one of the most valuable nuclear
stains known to the histologist, and is widely used as a chromatin
stain by the cytologist. On the other hand it has been used by
Galeotti as a cytoplasm stain following acid fuchsin and picric
acid; (see Krause, 1926-7, p. 1417). In the Ehrlich-Biondi technic
it is used to stain nuclei in contrast to acid fuchsin; (Id. p. 457);
while Bensley employs it to stain chromatin in contrast to acid

"This ordinarily occurs in trade as a zinc chloride double salt.

Phenyl Methane Dyes 163

fuchsin which stains the mitochondria; (see Lee, 1937, p. 166). It
is an ingredient of the Ehrlich triacid mixture (with orange G and
acid fuchsin) for staining blood smears. Botanists find it a
valuable stain, combined with acid fuchsin, for lignified xylem.
One of its most valuable uses today is in the Pappenheim (1899)
stain, in which it is combined with pyronin and used for staining
the gonococcus and mast cells as well as by Unna in studying
chromolysis. It is also a useful chromatin stain for protozoa, and
is employed in weak acetic acid solution for staining fresh ma-
terial beneath the cover glass.

When the foreign supply of dyes was first shut off, this stain
proved one of the most difficult to obtain in satisfactory quality,
largely due to the looseness with which the seventh methyl group
is attached and the resulting instability of the compound. At first
certain green dyes of an entirely different nature were furnished,
but as soon as an investigation of the dye was begun manufac-
turers proved perfectly able to produce methyl green; the difficulty
came in obtaining the right degree of purity. Samples were finally
furnished so pure that they lacked completely the necessary meta-
chromatic staining quality; and it proved necessary to add a certain
small percentage of the violet dye to obtain the proper results.
This problem seems to have been solved at present and satisfactory
methyl green is available. The chief problem now is to standardize
it. With other stains this can ordinarily be done on the batch
basis, approving some batch large enough to meet the demand for
a period of years. With methyl green this cannot safely be done,
on account of its instability. Hence large batches are impractical;
and the stain ought to be sold with the caution that the dye does
not keep indefinitely without change.

This instability complicates standardization by spectro tests.
Fig. 19, p. 145, shows a spectrophotometric curve of this dye in
comparison with those of light green SFY and fast green FCF.
The three are much alike with two maxima at about 430 and 630.
All the green dyes show disagreement between dye content deter-
minations by means of TiCls titration and comparisons of the
density at the peak of the chief maximum. Methyl green has an
additional source of inconsistency in its assay: the ratio between
two points of the curve equal distances each side of the maximum
fluctuates, due apparently to the fact that solutions change on
standing.

There seems, since 1930, to have been a change in the nature
of the methyl green samples supplied as biological stains. One of
the companies admits that its methyl green is not C. I. No. 684,
but the following:

164

B iolog ica I Stains

n42

ETHYL GREEN

Synonym: Methyl green*

c. I. NO. 685

CH, CI

N_.

-C

Br CH.CH,

1/
_N— CH3

\
CH,

CH,

N

CH,

CH,

C27H35N3CI Br; Mol. Wt. 516.947
{A basic dye)

This dye differs from the preceding only in that it is prepared
from crystal violet by the action of an ethyl instead of a methyl
halide. It is almost, if not entirely, indistinguishable from C. I.
No. 684 by spectrophotometric tests, and apparently acts very
similarly for biological purposes. It is entirely possible that it is
even better than the other for use in staining; certainly the present
methyl greens are more satisfactory than those on the market be-
tween 1920 and 1930.

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH EITHER
METHYL GREEN OR ETHYL GREEN IS USED

NAME OF PROCEDURE

Bismarck brown with methyl green in animal histology
Methyl green in glycerin jelly for pollen grains
Albert's stain for the diphtheria organism
Pappenheim-Saathof stain for gonorrheal pus
Saathof stain for bacteria in tissue

page reference to
Staining Procedures

IA4-5

nA-13

IIIA2-II

IIIA2-I9

IIIB3-5

si5

IODINE GREEN

C. I. NO. 686

This dye is closely related to methyl green, the generally accept-
ed formula being:

"This ordinarily occurs in trade as a zinc chloride double salt.

Phenyl Methane Dyes 165

CI CH3

CH3 CI

\l

N=/~'\=C

/ \^/
CH3

C27H35N3CI2; Mol. Wt. 472.488
{A basic dye)

Iodine green is a nuclear or chromatin stain which has selective
properties that make it of value in certain special procedures. It
was used by Ciaccio (1906) for staining nervous tissue, in combina-
tion with acid fuchsin and picric acid; by Lefas (see Krause, 1926-
7, p. 1122) as a blood stain in combination with acid fuchsin; and
by others as a stain for mucin and amyloid which it colors red
instead of green. It was employed by Zimmermann (1893) with
basic fuchsin for staining chromatin in plant tissue; while together
with acid fuchsin it has been proposed by other botanists for stain-
ing lignified xylem. Torrey and Buckell (1922) have recom-
mended a differential medium for the isolation of the gonococcus
which includes this dye.

ACID VIOLET

There are various dyes on the market known as acid violet
with some shade designation. These dyes vary extremely in
composition and they are listed under various Colour Index
numbers. They are in general sulfonated violet dyes of the rosan-
ilin group, some of them simple methylated pararosanilins, others
benzylated compounds.

Some acid violet — its exact identity uncertain — has been em-
ployed by Bailey (1921) in cytological studies on the human
pituitary gland; also by Maurer and Lewis (1922) for staining simi-
lar tissue from the pig; by Ono (1934) for staining spirochaetes in
blood, and by Weiss for staining both spirochaetes (1929) and
bacterial flagella (1928). It is very unfortunate that any biologist
should have been furnished a stain labeled merely acid violet; the
term is too indefinite for identification.

BENZYL VIOLET

There is a group of dyes known as benzyl violets, which are
pararosanilins with benzyl substitution in one or more of the

166 Biological Stains

amino groups. Some of them are acid and some basic dyes.
There is occasional reference in the biological literature to the use
of a benzyl violet, but without any indication as to which of the
dyes in question is intended.

n50

SPIRIT BLUE C. I. NO. 689

Synonyms: Geniiana blue 6B. Anilin blue, alcohol sohible.
Light blue. Lyon blue. Paris blue.

This is a mixture of di-phenyl rosanilin chloride:

H

I
N /-\

CI

H \/— \ NHj

CH3

C32H28N3CI; Mol. Wt. 490.025-

and tri-phenyl pararosanilin chloride:

CI

I

I \_/
H \/-\ N_/-\

"I \_/
H

C37H30N3CI; Mol Wi. 552.091

{Basic dyes absorption maximum of spirit blue 2R
about 581 in alcohol.)

Solubility at 26° C: in water nil; in alcohol 1.10%

Lynch (1930) mentioned Lyons blue (undoubtedly this dye) as a
satisfactory counterstain to carmine in bulk staining of protozoa
and other small animals and embryos. It has, moreover, been re-,
ported by others as a good contrast stain for carmine, bringing
out nerve fibers well in embryonic tissue; for this purpose it is
used in strong alcoholic solution. Quite recently, Knaysi (1942)
has mentioned this dye as one whose color base can be used in de-
termining hydrolysis of fats.

Phenyl Methane Dyes 167

N.55 VICTORIA BLUE 4R C. I. NO. 69O

Synonym : Fat bine IfR.

I
CH3 CI /— \ N CH3

CH. \_/-N /-\

CH3
C34H34N3CI; MoL Wt. 520.093
{A basic dye; absorption maxima 593.5 [538.5])
Solubility at 26°C: in water 3.23%; in alcohol 20.^9%

Herzberg (1934) suggested this dye for staining minute granules
in the case of certain virus diseases, claiming these granules to be
the filterable virus itself. Others (e.g. Gutstein, 1937) have fol-
lowed the same technic, staining the "elementary bodies", but
without committing themselves to Herzberg's theory. Isada
(1938) mentioned this dye as one of several that may be used in
staining bacterial flagella. Lipp (1940) has proposed it, either
alone, or mixed with methyl green and pyronin, for staining spiro-
chaetes, particularly Treponema pallidum.

The various Victoria blues are often confused; see also Victoria
blue R and B, a few pages below.

n60 methyl blue C. I. NO. 706

Synonyms: Cotton blue, Helvetia blue.

H

I
H /'~\=N_/~\_S03 -Na

I II \_/ I V-/

NaS03_/^\_N_/— \_C SO3

H

1

C37H27N309S3Na2; Mol. Wt. 799.784
(An acid dye; absorption maximum about 607)

On account of the sulfonic groups, this dye is strongly acidic
and makes a good counterstain. It may, for instance, be substi-

168 Biological Stains

tuted for anilin blue W.S., as in the Mallory connective tissue stain.
Such uses of this dye are too numerous to Hst, but one might men-
tion that of Dubreuil (see Krause, 1926-7, p. 1382) who employed
it, cpmbined with picric acid, in contrast to a red nuclear stain
such as carmine or safranin. One of its oldest and best known
uses is that of Mann (1894) who mixed it with eosin and showed
the value of the mixture in staining nerve cells. Recent workers
suggest it for other purposes: Clauser and Strani (1930) for stain-
ing unfixed tissue; Cumley (1935) for the negative staining of
bacteria; Monne (1935) in the vital staining of protozoa.

For use in buffered Mann stain, see Staining Procedures p. IA4-I9; in technic
with Biebrich scarlet see p. IB3-I2.

n65 anilin blue, w. s. (i.e., water soluble) c. i. no. 707

Synonyms : China blue. Soluble blue 3M or 2R. Marine blue V.

Cotton blue. Water blue.

This is a mixture of the tri-sulfonates* of tri-phenyl para-
rosanilin (C. I. 706) and of di-phenyl rosanilin. The latter is:

H

NaSOj /""\=N_/— \_S03-Na

H2N_/— \_C SO3

CHj

H

N
C32H25N309S3Na2; Mol. Wt. 737.718

{An add dye; absorption maximum of samples submitted for
certification 595-610 in alcohol)

Anilin blue W. S. should be regarded as a group of dyes rather
than as a simple dye. The composition of the various commercial
products sold under this name is uncertain, and no method seems
to be known for controlling the process of manufacture so as to
yield a constant product.

The spectrophotometric curve of a sample typical of those sold
as biological stains is given in Fig. 22.

Certain dyes of this group (apparently only the rosanilin deriva-
tives, because they are most likely to be free from undesirable
colored impurities), have indicator properties, decolorizing almost
completely on addition of alkali, and slowly becoming blue again

*The location of the sulfonic groups is uncertain.

Phenyl Methane Dyes

169

.6-

o
M

.5-

O

O

-J

.4-

>.

h-

.3-

(n

z

UJ

.2-

QC

1 -

O

. 1

«J

o

o

t

400

460 520 580 640
WAVELENGTH mu

Fig. 22. Spectral curve of anilin blue W.S,

700

if the reaction becomes acid. It is not a specially valuable in-
dicator, however, partly because of the slowness with w^hich it re-
sponds to change in reaction, and partly because it is quite subject
to protein and other errors. In spite of these drawbacks it has
sometimes been used in bacteriological media, when accuracy is
not important and the slow response to acid production is of no
significance.

Hendrickson, Baldwin, and Riker (1934) have employed it in
bacteriological media for a different purpose, the isolation of the
crown gall organism ; in their medium it holds fungi in check while the
desired organism absorbs the blue color and is thus easy to recog-
nize. For some reason not yet explained, those samples (apparent-
ly pararosanilin derivatives) which do not decolorize properly in
alkaline solution, fail to work in this medium, although the dye
does not serve in this instance as an acid-base indicator.

By far the best known use of anilin blue, W.S., is as a counter-
stain in histology. Among its histological applications are: by
Stroebe and Huber (see Krause, 1926-7, p. 63) as a cytoplasm
stain preceding safranin; by Galli for axis cylinders; frequently by
botanists as a contrast for safranin in vascular plant tissue, or for

170 Biological Stains

erythrosin or phloxine in algae; and very widely by pathologists in
the Mallory (1900) connective tissue stain, in which it is combined
with orange G and acid fuchsin; by Unna in contrast to orcein for
staining epithelial sections (see Lee, 1937, p. 438) and in studying
the process of chromolysis; by Koneff (1936) in combination with
iron hematoxylin as a polychrome staining method for routine
laboratory use.

On account of the lack of constancy in this group, anyone
publishing a technic calling for one of these dyes should be very
careful to give all the information obtainable from the label and
should specify the source from which the sample used was ob-
tained. If a Commission certified stain is employed, its certifi-
cation number should of course be given.

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH THIS STAIN IS USED

PAGE REFERENCE TO

NAME OF PROCEDURE

Staining Procedures

Mallory's anilin blue collagen stain

IB3-3

Heidenhain's "azan" modification

IB3-4

Grossman's modification

IB3-5

Acid alizarin blue modification

IB3-6

Biebrich scarlet and picro-anilin-blue in animal histology

IB3-I2

Lillie modification of Gallego elastic tissue stain

IB3-I5

Masson's trichrome stain

IB3-I8

Lillie modification

IB3-I9

Safranin and picro-anilin-blue in plant histology

IIA-11

Safranin and anilin blue in plant cytology

IIB-12

n70 ISAMINE blue C. I. NO. 7IO (?)

Occasional reference is made, especially in the European litera-
ture, to a dye called isamine blue. Its exact chemical nature is
not at present known, although the Colour Index Hsts isamine blue
6B or 8B as a synonym of No. 710 (alkah blue XG) and isamine
blue B and R as similar to this dye. The dye is not yet manu-
factured in America but was available from German sources be-
fore the second World War. It is known to be an acid dye, a
sulfonated naphthyl-rosanilin or naphthyl-pararosanilin.

Isamine blue is employed to some extent in Europe as a vital
dye by intravenous injection, followed by lead salts or radiation.
Exact references to such uses, however, are not now available.
Dean (1937) employs it for determining the proportion of antigen
to antibody in immune sera.

n75 patent blue V c. I. NO. 711

Synonyms: Aljphazurine 2G. Patent blue VF. Pontacyl

brilliant blue V.

Phenyl Methane Dyes 171

Usually the calcium salt of the following:

C,H=

/
N

SO3 CoHi

N_/-\_C /
/ \_/ \/-\_S03H
C2H5 \_/

\
OH

(C27H3xNAS2)2 Ca; Mol. Wt. 1159.388
{An acid dye)

This dye has rarely been mentioned in biological literature; but
is included by Ono (1934) among those employed for staining
spirochaetes; it is mentioned by McMaster and Parsons (1938) as
a vital stain, and by Yoe and Boyd (1939) as an acid-base and oxi-
dation-reduction indicator.

n80

XYLENE CY.

A.NOL FF

c. I. NO. 715

Synonym : Cyanol FF

CH3 C2H5

2H5

\

N / \ C

H

/ /

/ \ N

\ / / \
SO3 H

: /

\/ \ S03-Na

CH3 \

OH

C25H27N207S2Na; Mol. Wt. 554.599

{An aci

d dye)

Chambers (1935) has included this among several dyes which he
studied in connection with the uptake of dyes by cells in tissue cul-
ture. A cyanol, probably this one, has been used by Fautrez
(1936) in histochemistry, as an intracellular indicator; and by
Ivanov and Braun (1938) in the study of permeability of tissue
membranes.

c. Hydroxy Tri-phenyl Methanes (Rosolic Acids)

The rosolic acid dyes, as stated above, are tri-phenyl methane
derivatives in which the amino groups of the rosanilins are re-
placed with hydroxyl groups, thus giving them acidic instead of

172 Biological Stains

basic character. The compounds of this group are not very im-
portant as dyes and are scarcely used as stains. The greatest
interest of the biologist in them is due to their use as indicators.
In acid solution rosolic acid is yellow but it is converted by alkali
to the disodium salt which is red, thus:

0=/~\=C

pararosolic acid

0=

disodium pararosolate

There is considerable confusion in the nomenclature of these
dyes, as the names employed may be used in a strict chemical sense
or in a looser sense in practice. Chemically there are two rosolic
acids, which are related just as are rosanilin and pararosanilin.
Pararosolic acid differs from pararosanilin, only in having hydroxyl
groups in place of the amino groups:

H2N_/— \ /~\_NH. HO^/— \ /— \ OH

s /V
C

"\NH. / \/-X OH

H \_/ H

leuco-pararosanilin leuco-pararosolic acid

Rosolic acid, on the other hand, is a mono-methyl derivative, and
bears the same relation to rosanilin:

H,N_/— \ /— \_NH. H0_/— \ /— \ OH

\

CH3 / \/-\_NH, CH3 / \/-\_0H

H \_/ H \_/

leuco-rosanilin leuco-rosolic acid

Now the dye to which the name rosolic acic' " aurin is generally
given in practice is a mixture consisting of b< ih rosolic acid and
pararosolic acid together with other closely related compounds.
This dye is:

n90 aurin or rosolic acid C. I. NO. 72.4

The above names apply to the free acid. Yellow corallin is the
sodium salt.

Phenyl Methane Dyes 173

A mixture of rosolic acid and pararosolic acid, with oxidized and
methylated derivatives of the latter. This product is of consider-
able use as an indicator.

This dye was employed by Pappenheim (1898) in a decolorizing
solution following carbol fuchsin. The method was intended to
safeguard against confusion of the smegma and other acid-fast
bacteria with the true tubercle bacillus. After the usual carbol
fucnsin stain, the decolorizing was done with 1% rosolic acid in
absolute alcohol, saturated with methylene blue and containing
20% glycerol.

No other dyes of this group have biological use. Two others
perhaps deserve mention:

n93 red CORALLIN C. I. NO. 72.6

Synonym: Aurin R.

Probably a pararosolic acid salt of pararosanilin, forming an
intermediate stage in the conversion of pararosolic acid into
pararosanilin.

n95 chrome violet CG C. I. NO. 7I7

A carboxyl derivative of pararosolic acid :

COONa

/
/~\_0H

o_/--\_c

"\_0H

/ \_/

COONa \

COONa

C22Hii09Na3; Mol. Wt. 488.299

{An acid dye)

3. DIPHENYL-NAPHTHYL METHANE DERIVATIVES

P5 VICTORIA BLUE R C. I. NO. 718

Synonyms: New Victoria blue B or R. Corn blue B.

/CH3

CHj CI

/"

\_

N

\

_/

CH,

N_

=/"

~\^

_C

/

_/

~ \/"

~\

_NHCH2

•CH3

CHj

_/"

C29H32N3CI; Mol. Wt. 458.027

{A basic dye; absorption maxima 61Jf.7 [558])
Solubility at 26°C: in water 0.54.%; in alcohol 3.98%

174 Biological Stains

Some Victoria blue, possibly this or the following or possibly
C. I. No. 690, has been employed by Gutstein (1932) as a vital
stain for yeast, by Hausdorff (1927) for staining spermatazoa in
the testicle, and by Ono (1934) for staining spirochaetes in blood.
The various Victoria blues are frequently confused in the litera-
ture.

plO VICTORIA BLUE B C. I. NO. 72.9

Synonyms: Fat blue B. Com blue BN.

CHj

/
CH3 CI /~\_N_CH3

\1 /\ /

/ \_/ \/ \_NH_

CH,

/ \

C33H32N3CI; Mol. Wt. 506.067
{A basic dye; absorption maxima 619.2 [567])

This dye has been employed by Proescher (1934) for staining
neuroglia, and its eosinate by Geschickter (1930a) for fresh
frozen tissue.

Pll NIGHT BLUE C. I. NO. 73 I

CH2' CH3

-\_N/

/ \CH2.CH3

HN— \_/ I|/-X=N_CH2.CH3

CI CH2.CH3

C37H40N3CI; Mol. Wt. 562.171

(A basic dye)

Hallberg (1946) recommended this dye in place of carbol fuchsin
as a stain for acid fast bacteria, with phenolic pyronin as a counter-
stain. The procedure has been followed to some extent in Europe,
but is not much used in America. The dye has recently become
hard to obtain commercially.

Phenyl Methane Dyes

175

Pl5

Synonyms : Wool green BS, BSNA or C. Lissamine g
Pontacyl green S. Calcoid green S extra. Acid green

green B. Fast light green.

c. I. NO. 737

WOOL GREEN S y.. x. x-nw. /^/

BS, BSNA or C. Lissamine green By BS.
i green 8 extra. Add green S. Cyanol

NaSOs
C27H25N207S2Na; Mol. Wt. 576.603

(An acid dye; absorption maximum 63 Ji)

This dye has been mentioned by Lillie (1940, 1945) as a satis-
factory substitute for fast green FCF in his modification of the
Masson trichrome stain.

For use in Lillie modification of Masson trichrome stain, see Staining Procedures,
p. IB3-I9.

CHAPTER VIII

THE XANTHENE DYES

THE group of compounds known as xanthene dyes com-
prises a number of basic and acid dyes and quite a series of
indicators. In fact, the most valuable indicators known to
the chemist fall in this group. They are derivatives of the com-
pound xanthene:

O

C

/ \
H H

1. THE PYRONINS

The pyronins are methylated di-amino derivatives of xanthene.
They are closely related to the diphenyl methanes and are some-
times classed with them, as they have a carbon atom attached to
two benzene rings, and show the same tendency toward quinone
structure. Their formula, on the other hand, is like that of the
oxazins except that the nitrogen of the central ring is replaced by
a methenyl (CH) radical. Like the oxazins, the atomic grouping
may be assumed to be in either the paraquinoid or the ortho-
quinoid form, thus:

H.N

_0_

I

H

H

/
N— H

I
CI

H.N

or

CI

I

"C=

I

H

NH,

Another arrangement of the atoms is possible in which no quinoid
ring exists, namely :

H,N

CI

I
_0_

~c~

I

H
177

NH.

178

Biological Stains

This latter form might also be assumed for the oxazins and thiazins
as well, and this type of formula is frequently used for the azins;
but the xanthene dyes are more often represented in this form. If
this formula is adopted the quinoid ring cannot be accepted as
their chromophore. For this reason one of the quinoid formulae
seems preferable; and for the sake of uniformity the paraquinoid
form will be given in the following pages. It must be remembered,
however, that the other formulae are equally admissible; and it is
possible that the compounds occur in two or even all three of the
different forms.

q5

PYRONIN Y

Synonym: Pyronin G.
CH3 CH3

N_/\_0_/\^N— CH3

C. I. NO. 739

CH3

CI

\/ c \/

H
C17H19N2OCI; Mol. Wt. 302.795

{A basic dye; absorption maximum about 552)
Solubility at 26''C: in water 8.96%; in alcohol 0.60%

This dye, finding occasional application as a biological stain in
pre-war days, was not manufactured in America until 1931. For
most purposes pyronin B may be substituted for it; but Scudder
(1931) finds it necessary in her combined Gram-Pappenheim stain.

A typical spectral curve of this dye is given in Fig. 23, in which
it is compared with pyronin B. This graph, together with the
others in this chapter, is from Stotz et al. (1950) in which the spec-
trophotometric characteristics of the xanthene dyes are discussed.

qs

ACRIDINE RED 3B

C. I. NO. 740

This dye, in spite of its name, is not an acridine derivative, but
a pyronin, a lower homolog of pyronin G or Y:

-NH— CH3
CI

CH3— HN_/\_0_/^

"c"

I
H

C15H15N2OCI; Mol. Wt. 274.743

{A basic dye; absorption maxima: [508] 5Jli7.5)

Xanthene Dyes

179

490

I

510

I I 1 I 1

530 550

Tnr\/x

570

-1
590

Fig. 23. Spectral curves of two pyronins:
1. Pyronin B. 2. Pyronin Y.

It is rarely called for in biological work, but has been used by
Gomori (1943), mixed with methyl green, in a procedure for stain-
ing tissue to demonstrate deposits of calcium salts and sites of
phosphatase activity.

180 Biological Stains

%io

PYRONIN

li

C. I. NO. 741

CH3CH2

CH.CHs

\

N

/ "
CH3CH2

/\

1

/\

/
N— CH.CHi

CI

\/ c \/

H

C21H27

N.OCl; Mol. Wt. 358.899

(A basic dye; absorption maxiinum about 55'>)

This dye differs from pyronin Y only in that it is an ethyl in-
stead of a methyl derivative. As a result it is very slightly deeper
in shade but has almost identically the same staining behavior.
The two dyes have very similar spectral curves, as shown by the
graphs in Fig. 23.

The pyronins find their principal use in the Pappenheim (1899)
combination, where a pyronin is employed with methyl green for
staining basophile elements, especially the mast cells, and for stain-
ing the gonococcus in smears of pus. It is also used sometimes as a
counterstain in the Gram technic for bacteria (see p. 160); and by
Ehrlich and Lazarus (1898) as a component of certain "neutral"
stains. Monn^ (1938) has employed a pyronin for staining mito-
chondria; while Huddleson (1931) makes use of its bacterio-
static properties in distinguishing between species of the bacterial
genus, Brucella.

For technic of Pappenhelm-Saathof stain for gonorrhoeal pus, see Staining Pro-
cedures,* p. IIIA2-I9.

For technic of Saathof stain for bacteria in tissues, see Id. p. IIIBj-o.

2. THE RHODAMINES

The rhodamines are similar to the pyronins except that there is
a third benzene ring attached to the central carbon atom arid
attached to this ring is a carboxyl group in the ortho position.
This latter group, although of acid tendency, does not counteract
the basic action of the amino groups, so the dyes are basic in
character.

♦Conn and Darrow (1943-4).

q20

Xanthene Dyes

RHODAMINE B

181

c. I. NO. 749

Synonyms: RJiodamine 0. Brilliant pi?ik B.

CH3CH,

CH.CH,

N

/\

/\

CH3CH3

/
N— CH.CH3

\/

c

1

X/

CI

/\

COOH

C28H31X2O3CI; Mol. Wt. 479.001
{A basic dye; absorption maxima about 556.5, [517])
Solubility at 26°C: in water 0.78%; in alcohol 1.^7%

A rhodamine, probably the above dye, has been used by Gries-
bach with osmic acid to fix and stain blood simultaneously; by
Ehrlich as a component of *'neutral" stain mixtures; also for his-
tological work in contrast to methylene blue; and by others
in contrast to methyl green. (See Krause, 1926-7, p. 2023.) Al-
though these early users were not always careful to specify which
rhodamine had been employed in their work, rhodamine B has
been definitely specified by later workers, e.g.: by Houcke (1928)
as a stain for glandular tissue, when mixed with methylene blue;
by Strugger (1938) as a vital stain for Allium epidermis, calling
attention to its fluorescence under ultraviolet light; by Monne
(1939) in studies on the Golgi apparatus; by Levaditi et al. (1940),
as a fluorochome of value in virus studies; by Metcalf and Patton
(1944), as a fluorochrome for use in insect histology; and by Fred-
erick (1941), as a microchemical reagent for the estimation of
antimony. It seems to be coming into more common use at pres-
ent, thanks in good part to its fluorescent properties in ultraviolet
light.

As a rhodamine is not infrequently specified without a shade
designation, it is sometimes not possible to be sure that the above
dye is intended. If not, the following is the dye most probably
indicated.

182 Biological Stains

q25 RHOD AMINE 6g C. I. NO. J ^2.

Synonyms: Rhodamine 6GX and 6GDN extra.
Calcozine red 6G extra.

By esterification of a compound closely related to rhodamine B,
the following is obtained:

CH3 • CH2— HN_/\_0_/\_NH— CH2 • CH3

\
CI

"C=

COOCHoCHa

C26H27N2O3CI; Mol. Wt. 450.949
{A basic dye; absorption maxima [492] 52&)

This dye has not been specified as frequently as rhodamine B,
but is mentioned by Strugger and by Monlie (cited above), each
of whom employed both dyes. Whether this ethyl ester has any
advantage over Rhodamine B is not stated.

A somewhat different dye, known as Rhodamine S (C. I. No.
743) has been mentioned in the same connection and may have
been used for some of the above-mentioned purposes. It is not a
true rhodamine, however, but belongs to a closely related group of
compounds, the succineins; for it does not have the three benzene
rings, the radical C6H4 • COOH being replaced by C2H4 • COOH.

q26 VIOLAMINE R C. I. NO. 758

Synonyms : Acid violet JfR. Fast acid violet SRL, A2R and R.

Na-S03_/-\_ /l |_ =1 1 N

NH V/ C \,

/ / \

CH3 /\_COONa CH3

C34H2406N2SNa2; Mol. Wt. 634.608
(An acid dye)

Lillie (1945 c) has mentioned this dye, among others, as a stain
for collagen in tissue sections.

Xanthene Dyes 183

q30 FAST ACID BLUE R C. I. NO. 760

Almost the same as: Violamine SB.

N_/"~\_0 CH. CH3

CseHaeNaOgSNazCU; MoL Wt. 763.552

{An acid dye; absorption maximum 53^)

The dye indexes (both Schultz and the Colour Index) list vi-
olamine 3B as almost synonymous with this. Violamine 3B, how-
ever, is a mixture containing a small amount of some red dye.
Some samples of fast acid blue R, made for textile application, are
also mixtures, although pure samples have been obtained. It is
hoped that pure dye of this type may become regularly available
for use as a biological stain.

This dye has been recommended by Romell for use in phenolic
solution for staining bacteria in soil. In the case of soils (mucks,
forest soils, etc.) containing much brownish or reddish matter,
this blue dye gives greater visibility of the microorganisms than
dyes of the eosin series.

In practice the rhodamines are prepared not from xanthene but
by the condensation of two molecules of dialkyl meta-amino
phenols,

R2N

/ \_0H

\_/

with one of phthalic anhydride:

CO

\
O

. /
CO

This shows their close relation to the next group of dyes, namely
the fluoran derivatives, which as will be seen are also prepared
from phthalic anhydride. In fact these two groups of dyes, acid
and basic respectively, are related in exactly the same way as the
rosolic acids and the rosanilins, the one group having hydroxyl
radicals where the other has amino groups.

184 Biological Stains

3. FLUORAN DERIVATIVES

Fluoran is not a dye, but is a very important compound in dye

chemistry. It is a derivative of phthalic anhj'^dride, and contains

a xanthene ring (five C atoms and one O atom) as well as a lactone

ring (four C atoms and one O atom) besides three benzene rings;

thus:

O

The fluoran dyes are derivatives of this by the introduction of
hydroxyl groups into two of the benzene rings at the para position
to the central carbon atom and the further introduction of halogen
atoms at various positions in all three benzene rings.

It proves convenient here to class these compounds with the
xanthene dyes. They may, however, be equally well considered
tri-phenyl-methane dyes, as can be seen by a glance at the formula
of any of them; in fact they are generally so considered by the
chemists. To the biologist they stand in a distinctly different class
from the tri-phenyl-methanes; and for that reason are treated here
instead of in the preceding chapter. The dyes of this group are
also, and equally correctly, spoken of as the fluorescein derivatives,
fluorescein being, as will be seen on p. 186, a salt of di-hydroxy-
fluoran. Sometimes, moreover, the fluorescein dyes are referred
to as the eosins. This term to cover the whole group is hardly
justified, as it is better to call eosins only the dyes definitely so
named below.

The fluorescein dyes are all similar in their action, but with
certain decided differences. In order for the reader to obtain a
real understanding of these stains and their behavior, a general
discussion of the whole group is necessary.

The dyes in this group most important to the biologist are:
eosin Y, C. I. No. 768 (also called eosin yellowish or eosin water
soluble); ethyl eosin, C. I. No. 770 (also called eosin alcohol
soluble) ; eosin B, C. I. No. 771 (also called eosin bluish) ; erythro-
sin B, C. I. No. 773; phloxine, C. I. No. 774; rose bengal, C. I.
No. 779. It is not yet absolutely certain which of these dyes is
most suitable for any particular purpose; but some information
is available on the subject. The difference in behavior of the
different dyes of this group seems to depend upon two factors:
difference in color and difference in acidity. The relation between
these two factors and chemical structure was discussed by Conn

Xanthene Dyes 185

and Holmes (1926) ; by reference to which paper it will be seen that
in color the dyes above listed increase in depth in the following
order: eosin Y, ethyl eosin, eosin B, erythrosin B, phloxine, rose
bengal. In the case of each of these, the color is lighter or deeper
according to the number of halogen atoms present. Eosin B is
the strongest acid in the group, and eosin Y the next strongest;
erythrosin is weaker, phloxine weaker than eosin and possibly
weaker than erythrosin; while rose bengal is the weakest of all.

To interpret this information into terms of actual use, it is neces-
sary to remember that there are two main types of histological pro-
cedures in which eosin is used: first as a general counterstain
(usually in alcoholic solution) following a basic dye; secondly, as a
cytoplasm stain (always in aqueous solution) preceding a basic dye.
In the first of these types of procedure it is important that the
dye be one with diffuse staining properties and with a color show-
ing good contrast to the basic dye employed (generally methylene
blue or hematoxylin). The more acid and lighter colored dyes in
the series (eosin Y, ethyl eosin, and eosin B) seem to possess these
properties to the greatest extent and accordingly to be best adapt-
ed to such procedures. The particular dye to choose depends
undoubtedly on the exact shade desired.

The second type of procedure (i.e. preceding a basic dye) is rep-
resented by the Mallory phloxine-methylene-blue stain and the
Held erythrosin-methylene-blue technic. In such procedures as
these, both dyes are used in aqueous solution; and there is there-
fore much more opportunity for chemical interaction between the
acid dye already in the tissue and the basic dye subsequently
added than when the acid dye is used in alcoholic solution. (It is
well known that acid and basic dyes in alcoholic solution do not
form insoluble reaction products.) In procedures like the Mallory
technic, there seems to be a tendency (possibly on account of this
interaction) for the acid dye to stain the cytoplasm too weakly
when followed by a basic dye, in case the very acid eosin Y is used.
Thus Held employs erythrosin, and Mallory who formerlyjspeci-
fied eosin discovered later that his original eosin was not a true
eosin Y, phloxine giving better results in his technic than one of the
true eosins. Now phloxine and erythrosin are not only deeper in
color than eosin Y, but are also less strongly acidic; and it is pos-
sible that their chemical nature rather than their color may be the
decisive factor in determining their superiority for such pro-
cedures as this. Rose bengal is even deeper in color, and is still
less strongly acidic; in fact, it seems to be too purple to contrast
well with methylene blue and of such a weakly acid character that
it tends to remain in the nuclei when used by the technic in ques-
tion. It might prove valuable preceding some basic dye; but it
has been found to give poor results preceding methylene blue in
the Mallory technic.

186

Biological Stains

In the procedure for staining bacteria in soil, as developed by
Conn (1918) and by Winogradsky (1924), the matter is still
further complicated by the fact that the dye must be of such a
color and such a strong acid as to stain the bacteria, but not the
dead organic matter present.

Another factor of much importance in the staining action of
these dyes is the amount of mineral salt present. It has, in fact,
been shown by Conn and Holmes (1928) that the intensity with
which a dye of this group can stain bacteria may be greatly in-
creased by adding a minute amount (0.001 to 0.1%) of some
mineral salt such as CaCl2. This matter is discussed in Chapter II.

There has been in the past much mislabeling of the dyes of this
group. Thus it is evident that the eosin used by Mallory when he
first worked out his eosin-methylene-blue technic must have been
a phloxine or some closely related dye rather than a true eosin.
There is evidence that erythrosin B and phloxine have been put on
the market labeled eosin bluish; so has a blend of eosin Y shaded
with some one of the higher members of the group. Phloxine has
been sold as magdala red and is even now sometimes marketed
under that name both by American and foreign concerns. All
this has caused much confusion; but dealers in stains are using
greater caution in the matter at present.

Rl

FLUORESCEIN

C. I. NO. 766

This is the simplest of the fluoran dyes, and is the mother sub-
stance of the eosins. Its sodium salt is known as uranin.

HO

O

OH

NaO

O

-0

O

_l
CO

COONa

C20H12O5; Mol Wt. 332.296
Fluorescein

C2oHio05Na2; Mol. Wt. 376.274
Uranin

{An acid dye: absorption maximum about 490)

Solubility at 26°C:

Of fluorescein: in water ^ 0.03%; in alcohoU 2.21%.
Of uranin: in watery 50.02%q; in alcohol^ 7.19%o.

Xanihene Dyes 187

Fluorescein is a yellow dye of very low tinctorial power, and
hence of no value for ordinary staining purposes. It is on the
other hand extremely fluorescent, the greenish yellow fluorescence
being detectable in extremely high dilution. On account of this
latter property the dye is used to determine the possibility of con-
tamination from some suspected source getting into a neighboring
water supply.

This fluorescence is even more pronounced in ultraviolet light, a
fact which makes the dye useful in fluorescence microscopy. Thus
Hercik (1939) employs it as a fluorochrome in the study of epi-
dermis cells of Allium; Levaditi et al. (1940) in virus studies; Met-
calf and Patton (1.944) in insect entomology.

It has been included by Williams and Green (1935) in a series of
dyes that prove useful in media in which fungi are growing, the
absorption of the dye by the fungi giving characteristic colors to
the colonies.

«« EOSIN Y (i.e., yellowish) c. I. NO. 768

Synonyms: Eosiriy water soluble. Bromo acidy J, TS, XL or XX.
Bromofluorescein. Bronze bromo ES.

Various shades denoted: Eosin B extra, BP, BS, DH, G, GGF, J
extra, SJ, JiJ, JJF, KS, S extra, Y extra and YS.

This dye is typically tetrabromo fluorescein:

Br Br

NaO I I

_Br

'\/-c=\/

Br I

i I COONa

C2oH605Br4Na2; Mol. Wt. 691.906
{An acid dye; absorption maximum 515-518)
Solubility at 26°C: in water U-20%; in alcohol 2.18%

but the mono- and dibromo derivatives are also known and fre-
quently occur in eosin. This affects the shade, as the more bro-
mine present the redder the dye. It is plain that various mixtures
of these compounds are on the market; but it has not yet been de-
termined which are more suitable for biological purposes. Con-
siderably more work on eosin is needed than has been done at the
present time. From the name "water soluble eosin'* it is often

188

Biological Stains

assumed that this dye is not soluble in alcohol. This is not true,
however.

A typical absorption curve of eosin Y is given in graph 2, Fig.
24, in which it is compared with the similar spectral data for eosin
B and ethyl eosin.

Like fluorescein, eosin Y shows greenish yellow fluorescence,
especially in alcoholic solution. This fluorescence is visible in

.6-

9.5

>-.4

H

(/)

Z
UJ

o

O

-I

o
0.2

.1-

400

.3-

420

440

460

480 500

520

540

)"

Fig. 24. Spectral curves of three eosins:
Eosin B. 2. Eosin Y.

3. Ethyl eosin.

ordinary light, but although quite strong, "is not so pronounced nor
visible in such great dilution as in the case of fluorescein. It is
interesting that in this series of fluoran derivatives, tinctorial
power increases but fluorescence decreases with the ascending
members of the series; thus the erythrosins are less fluorescent than
eosin, while phloxine and rose bengal show scarcely any fluorescence
in ordinary light. In spite of the marked fluorescence of eosin Y,
it does not seem to have been suggested as a fluorochrome.

Xanthene Dyes

189

Yellowish eosin is one of the most valuable plasma stains known.
It is used in various technics for staining the oxyphile granules of
cells (i.e., the granules having special affinity for acid dyes); these
cell elements, in fact, being often called eosinophile granules be-
cause their presence was first recognized through the use of this
dye. It is often employed as a counterstain for hematoxylin and
the green or blue basic dyes; as for example by List with methyl
green. Its uses, however, are really too numerous to list. At
the present time one of the uses for which it is in greatest demand
is as a blood stain in the technic of Romanovsky, with its various
modifications, in which it is combined with methylene blue to form
a "neutral" stain.

A dye mixture sold under the name of "Triosin", was described
by Galigher (1934), and sold by him without stating its composi-
tion. The mixture proves valuable as a counterstain in some pro-
cedures where eosin Y alone gives less satisfactory results. Re-
cently Emery and Stotz (1952) used this product as an unknown
in a test of filter paper chromatography for dye analysis. In the
course of this investigation the composition of the mixture became
evident, and was stated by these authors as being apparently 62%
eosin Y, 28% orange G, and 10% erythrosin Y. A spectral curve
of the mixture, however, does not reveal the presence of either of
the two latter dyes, but seems practically identical with that of
eosin Y alone.

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH THIS STAIN IS USED*

NAME OF PROCEDURE

PAGE REFERENCE TO

Staining Procedures

Delafield's hematoxylin with eosin Y

IA4-II

Mayer's acid hemalum with eosin Y, Lillie modification

IA4-I3

Thiazin eosinates in tissue staining

IA4-I7

Buffered Mann stain

IA4-I9

Wright stain for blood films

ID3-3

MacNeal's tetrachrome stain for blood films

ID3-5

Giemsa stain for blood films

ID3-G

Wright, Giemsa, or May-Gruenwald stain for bone marrow

ID3-IO-II

Counterstain in Gram-Weigert technic for bacteria in tis-

sues

IIIB3-8

*Under this heading are given references to procedures described in detail in
Staining Procedures, edited by Conn and Darrow (1943-4).

r8

METHYL EOSIN

Synonym: Eosin, alcohol soluble.

c. I. NO. 769

This is the methyl ester of yellowish eosin, the sodium salt of
which is:

190

Biological Stains
Br Br

NaO

COOCH3

C2iH905Br4Na; Mol. Wt. 683.943
{An acid dye; absorption maxima about 520y [JfSS.d])

There is no evidence that this dye has been furnished to biol-
ogists as a stain. The alcohol soluble eosin known in the biological
laboratory is apparently the following.

Rio ETHYL EOSIN C. I. NO. 770

Synonyms : Eosin, alcohol soluble. Eosin S.

This is similar to methyl eosin, but is the ethyl ester:

Br Br

NaO

O

COOCHoCH,

C22Hii05Br4Na; Mol. Wt. 697.969
{An acid dye; absorption maximum 524--527)
Solubility at 26° C: in water 0.03%; in alcohol 1.13%
A spectral curve of this dye is given in graph 3, Fig. 24.

Ethyl eosin is apparently the alcohol soluble eosin ordinarily
sold by biological supply houses. For some time after the first
World War it was difficult to secure this dye in America, and one
stain company for a few years furnished the color acid of eosin Y
when alcohol soluble eosin was ordered. (The color acids of all
these dyes are alcohol soluble but almost insoluble in either hot or
cold water; methyl and ethyl eosin, on the other hand, are slightly
soluble in cold water but strongly soluble in hot water and alco-
hol.) This error has now been corrected, and true ethyl eosin
as obtainable from .American manuf acturer§, is on the Commission

Xanthene Dyes 191

certification basis, and can be ordered from any biological supply
house. It should be ordered as ethyl eosin rather than alcohol
soluble eosin, as the latter name is less definite.

Ethyl eosin is a valuable counterstain after Delafield's hema-
toxylin. Preceding methylene blue, it is employed in demonstrat-
ing Negri bodies in the central nervous system of rabid animals.

For technic of Harris stain for Negri bodies, see Staining Procedures, p. Ill A2-2O.
For technic of staining Negri bodies in sections, see Id. p. IIIB3-I2, 13.

Ri5 EOSIN B (i.e. bluish) c. I. NO. 771

Synonyms: Eosin BN, BAy BSy BW, or DHV. Saffrosin. Eosin
scarlet. Eosin scarlet B. Scarlet J, J J, F. Nopalin G.

Imperial red.

This is a dibrom derivative of dinitro fluorescein :

NaO

C=^

NO.

I I COONa
\/

C2oH6N209Br2Na2; Mol. Wt. 624.090

{An acid dye; absorption maximum 516-519)

Solubility at 26°C: in water 39.11%; in alcohol 0.75%

This dye has a distinctly bluer shade than eosin Y; although
its absorption maximum is not very different, it has a distinctly
different spectral curve, as shown by Fig. 24, p. 188.

It has occasionally been called for as a counterstain in some
histological procedure as, for example, in aqueous solution follow-
ing Mayer's hemalum (Kornhauser, 1930), but is not in general
very valuable. Lillie (1944c) finds it specially useful, as a neutral
stain, for tissues, as an azure A or toluidine blue eosinate.

Ordinarily, if a shade deeper than eosin Y is desired, better
results can be obtained with erythrosin, phloxine or rose bengal
than with eosin B. Coleman and Bell, in fact, has put on the
market a product labelled * 'eosin, bluish blend," which is a mixture
of eosin Y with some one of the dyes just named; it is very satis-
factory for certain staining procedures, but must not be confused
with true eosin B.

For technic of Mayer's hemalum with eosin B, see Staining Procedures,
p. IA4-I2.

For technic of buffered azure eosinate method, see Staining Procedures,
p. IA4-I2.

19'2 Biological Stains

r20 mercurochrome ixo

This is a proprietary name applied to a fluorescein derivative
closely related to eosin: dibromo-hydroxy-mercuri-fluorescein.

Br Br

NaO I

\/V_o_

J

C2oH806HgBr2Na2; Mol. Wt. 750.700

The dye has been widely advertised as a disinfectant especially
for the skin; and there has been considerable discussion in the
literature as to its actual value for the purpose. It has staining
properties not unlike an erythrosin, or phloxine. Baldwin (1928)
states that it may be used in 2% aqueous solution in place of eosin,
especially for blood woxk and for tissues after Zenker fixation;
thus employed it is more intense than eosin and has a stronger
affinity for cytoplasmic structures. Detwiler and McKennon
(1929) employ it in concentration of from 1 :500,000 to 1:1,000,000
as a fungicidal agent for the treatment of amphibian embryos.
Conklin (1934) recommends it as a counterstain to malachite green
in the Wirtz spore stain for bacteria.

R25 ERYTHROSIN, YELLOWISH C. I. NO. 77I

Synonyms: Erythrosin R or G. Pyrosin J. Dianthine G. lod-

eosin G.

This is a fluorescein in which there are two substituent iodine
atoms instead of four bromine atoms as in yellowish eosin.

I I

NaO I I

0_/\ O

c=

COONa

C2oH805l2Na2; Mol. Wt. 628.09S
{An acid dye; absorption maximum about 510.5)

Xanthene Dyes 193

r26 erythrosin, bluish c. I. NO. 773

Synonyms: Erythrosin B, N, or JN. Pyrosin B. Eosin J.

lodeosin B. Dianthine B.

This is the tetraiodo compound corresponding to the tetrabrom
compound of typical eosin.

NaO I I

. O /\_0

C=

I

COONa

C2oH605l4Na2; Mol. Wt. 879.922
{An acid dye; absorption maximum 52^-527)

w

Solubility at 26°C: in water 11.10%; in alcohol 1.87%

There is some uncertainty as to which of these two very similar
dyes is preferable in any given technic. At present, the latter,
erythrosin B, is ordinarily furnished by supply houses when
"erythrosin" is ordered. Its spectral curve is given in Fig. 25,
p. 194.

Erythrosin has some use as an indicator. It is also employed
as a contrast stain for hematoxylin and certain blue and violet
nuclear stains. Thus, for example, Held (1895) used it, preceding
methylene blue, as a plasma stain for nerve cells. Similarly the
technic of Jackson (1926) calls for it as a counterstain to crystal
violet in plant histology. It was employed by Winogradsky (1924)
for staining bacteria in soil. Gellhorn (1931) applied it to the
vital staining of sea urchin eggs. These procedures are mentioned
merely by way of illustration; the uses of erythrosin are quite
numerous, and the list could be extended almost indefinitely. For
these purposes probably the tetra-iodo compound (i. e., erythrosin
bluish) is desired; but the literature is vague on the subject.

A sample of erythrosin of pre-war origin that was labeled mag-
dala red has been examined by the Commission. This mislabeling
undoubtedly explains Chamberlain's results already mentioned
(page 133) in staining algae. Chamberlain, it will be recalled, was
able to obtain good results with a low-priced product called mag-
dala red but not with the high-priced stain called magdala red
echt.

For technic of Jackson stain for woody tissue, see Staining Procedures, p. IIA-9.

194

Biological Stains

.6-

r

0.5-

L.
460

480

I I I I
500 520

540

560

Fig. 25.
1. PhloxineB.

Spectral curves of three xanthene dyes:

2. Rose Bengal. 3. Erythrosin B.

If in the manufacture of fluorescein, dichloro or tetrachloro
phthalic acid is used for condensation with resorcin, a series of
halogenated derivatives may be obtained differing from those just
named by having halogen atoms in the phthalic acid residue of
the molecule as well as in the resorcin residue. These compounds
are slightly deeper in shade than the corresponding derivatives of
simple phthalic acid; and they are generally regarded as being of
more pleasing shade than the ordinary fluorescein dyes. The
phloxines and rose bengals are the best known of these dyes.

Xanthene Dyes 195

R30 phloxine

c. I. NO. 774

Synonyms : Erythrosin BB, or B extra. New pink.

Br

NaO

_0_

_>

Br

1

Br

CI

—

-c=

:

\/\

Br

"COOXa

\/

"CI

C2oH405Br4Cl2Na2; Mol. TT7. .760.804

(An acid dye; absorption maxima about 535.7, 14-97. 1])
Solubility at 26°C: in water 50.9%; in alcohol 9.02%

This dye and the following are often denoted interchangeably
phloxine or phloxine B, and there is some question as to whether
biologists have always been furnished the same type. The phlox-
ines of American manufacture at present on the market are the
following type.

RSI PHLOXINE B C. I. NO. 778

Synonyms: Phloxine TA, iV, BP super, RB, TB, or BB.

Cyanosine. Eosin 10 B.

C2oH205Br4Cl4Na2; Mol. Wt. 829.702
(An acid dye; absorption maxima 54-6-54-8)

This dye differs from C. I. No. 774 in having four instead of
two chlorine atoms in the phthalic acid residue of the molecule,
Samples of phloxine recently submitted to the Commission for
certification seem to vary in degree of bromination or chlorination,
but correspond to this C. I. number rather than to the preceding.
Although some of the procedures given below may have been
worked out originally with the other type of phloxine, this type
has been found to give satisfactory results.

A spectral curve of this dye, in comparison with erythrosin B
and rose Bengal, is given in Fig. 25.

Unna (1921) used phloxine in combination with several other
acid dyes in studying the process of chromolysis. The dye was
seldom specified for biological work until about 1925, but has re-
cently been applied to staining bacteria, Negri bodies, and to
various histological procedures. It has, moreover, frequently been
used under other names.

196

Biological Stains

Chamberlain (1932, page 69; 1927), mentions having used it
successfully in place of magdala red in staining algae. His original
technic called for magdala red ; but true magdala red does not serve
his purposes. Inasmuch as erythrosin (see above) was evidently
sold in the past as magdala red and Chamberlain can duplicate his
original results with phloxine, the chances are that some of the
magdala red formerly available was either phloxine or else that
phloxine and erythrosin give similar results by Dr. Chamberlain's
technic.

About 1920 Dr. F. B. Mallory, like many others, found it
difficult to obtain an eosin of either domestic or foreign origin
which would give consistently reliable results by his eosin-methy-
lene blue method described in 1904. After testing out, on the
advice of a member of the Commission, a series of eosins and
closely related dyes he wrote that phloxine is "the best eosin I
have yet found for use in the eosin-methylene blue stain for paraffin
sections of tissues fixed in Zenker's fluid."* Here again is a case
where phloxine apparently was obtained before the war under an
incorrect name and the incorrect name used in the publication of a
well-known technic.

For technic of Mallory's phloxine-methylene-blue stain, see Staining Pro-
cedures, pp. IA4-2O; IIIB3-4.

For use as counterstain in Gram-Weigert technic for bacteria in tissues, see Id.
p. IIIB3-8.

r35

C. I. NO. 777

ROSE BENGAL G

Synonym: Rose SA,
Various shades denoted: Rose bengal Ny AT, NTy NTOy and B.

NaO

0-/V=0

CI

c=

I

I I I

COONa

-CI

C2oH405l4Cl2Na2; Mol Wt. 948.820
{An acid dye)

This particular rose bengal apparently has not been employed
by biologists. The dye of that name sold by stain companies at
present is the following.

*Quoted from personal letter.

Xanthene Dyes 197

r36 rose bengal C. I. NO. 779

Various shades denoted: Rose bengal extra^ SB Conc.y N extra^ DYy
B and 2B.

I I

NaO I I

o_/\=o

C1~\/~C1

I

CI
C2oH205l4Cl4Na2; Mol. Wt. 1017.718
(An acid dye; absorption maximum S^^^^^-S)
Solubility at 26°C: in water 36.25%; in alcohol 7.53%

The dye has a pleasing deep pink color; and although an acid
dye it proves to have considerable affinity for bacterial protoplasm,
and to have good selective properties when used as a bacterial
stain. It has been recommended (Conn 1918, 1921) for staining
bacteria, especially in soil suspensions. It has also been used as a
cytoplasm stain following hematoxylin or preceding toluidine blue.
Maneval (1934!) employed it in the negative staining of bacteria
and Ono (1934) for spirochaetes in blood. It also finds use in
Delprat and Stowe's test (1931) for liver function. Popper (see
Metcalf and Patton, 1944) mentions it as a useful fluorochrome in
the study of fats under ultraviolet illumination. Smith and Daw-
son (1944) employ this dye as a bacteriostatic agent in media de-
signed to permit the growth of soil fungi while repressing the
bacteria; this is the only instance yet noted of one of the fluoran
derivatives being thus used.

From Fig. 25, in which the spectral curve of this dye and phlox-
ine B are compared, it will be seen that they differ more in shape
than in the position of the absorption maximum.

For technic of general staining of pure cultures of bacteria, see Staining Pro-
cedures, p. IIIA2-5.

For technic of Conn's stain for bacteria in soil, see Staining Procedures, P.IIIA2-
21.

4. PHENOLPHTHALEIN AND THE SULFONPHTHALEINS

A phthalein is a compound of phthalic acid :

COOH

COOH

198 Biological Stains

or rather of phthalic anhydride:

CO

\
O

/
CO

with phenol or a phenol derivative. If phthalic acid is heated
with phenol and sulfuric acid it combines with two molecules of
the phenol and forms phenolphthalein. In the same way, a sul-
fonphthalein is a compound of ortho-sulfo-benzoic acid:

/\_S03-H

"COOH

and phenol or a phenol derivative. These compounds, although
sometimes behaving as dyes, are not used as dyes or stains, but as
indicators. For this purpose the members of the group are very
valuable.

Phenolphthalein, although not used as a dye, is colored and is ap-
parently capable of salt formation. In acid solutions it is colorless,
and is assumed to have the formula:

HO OH

Upon neutralization the alkali is believed to attach itself to the
CO-group, which breaks the five-sided ring (the lactone ring) and
causes one of the benzene rings to take on quinoid form, thus:

HO /\ /\—0

COONa

With this change, the red color of the compound appears, but dis-
appears again if the solution is made acid so as to destroy the quin-
oid structure. This makes the compound a very valuable indi-
cator.

Xanthene Dyes 199

The dyes of this group have no value in practical dyeing, al-
though they are all weakly acid dyes. Similarly they are of little
use as biological stains, except occasionally in vital staining. Their
real value is as acid-base indicators. Even when they are em-
ployed as vital stains, they serve primarily to study the reaction of
tissues or body fluids. They are also valuable indicators in
micro-injection of cells.

Hydrogen-ion indicators. The sulphonphthaleins, together
with phenolphthalein, are among the most useful indicators known
to the chemist. Quite a long series of them has been prepared,
which in general show their deepest color in alkaline solutions and
turn yellow on the addition of acid. Some of them, such as
thymol-sulfonphthalein (thymol blue), show two colors besides
yellow, one in strong acid solutions and the other in strong basic
solutions, while in solutions near the neutral point they are yellow.
That these color changes are due to alterations in the structure of
the molecule, such as the disappearance and reappearance of the
quinoid ring, is generally assumed; but in the case, of these com-
pounds the relation of structure to color is complicated and has
not yet been worked out to general satisfaction.

The colorimetric measurement of H-ion concentration depends
upon these color changes. The color change of an indicator takes
place within a short range on each side of that point in the H-ion
scale at which the dye is 50% dissociated, i.e., occurs half in the
form of the undissociated dye and half in the form of free ions.
This point on the pH-scale (at which an indicator is 50% dis-
sociated) is important, and corresponds to the invert logarithm of
its dissociation constant. The dissociation constant of an in-
dicator, in other words, is a quantity whose invert logarithm is the
pH-value of the point at which the indicator is half dissociated.
The approximate invert logarithm of the apparent dissociation
constant is denoted by chemists by the symbol pK; the value of
this quantity is given in the case of each of the indicators listed
below.

For a short distance on each side of the dissociation constant,
every shade of the indicator corresponds to a definite pH-value;
this zone is known as the sensitive range of the indicator. Roughly
speaking, the sensitive range of any indicator extends for about
0.8 pH to each side of its pK- value. Throughout its sensitive
range, an indicator can be used to determine the H-ion concentra-
tion of a solution by comparing its shade with that produced in
standards of known reaction. The various indicators of this group
differ in their strengths as acids (i. e. the extent to which they dis-
sociate) ; and the greater their strength the lower the pH-value of
the point at which they are 50% dissociated. It is plain, therefore
that different indicators may be selected according to their dis-
sociation constants to cover successive portions of the H-ion scale.

200

Biological Stains

Fortunately nearly all of them have different dissociation con-
stants; so that by employing a series of them one may determine
the H-ion concentration of solutions of any reaction ordinarily
encountered.

There are two general types of indicators, those which show
only one color in the useful range, and those which change from one
color to another. The phthaleins, such as phenolphthalein, change
from colorless substances in their acid ranges to colored compounds
in their alkaline ranges. The sulfonphthaleins, on the other
hand, change from yellow to some deeper color. With the first

TABLE 3

COLOR CHANGES OF THE INDICATORS OF CLARK AND LUBS AND OF COHEN

CONCEN-

MOLECULAR

TRATION

FULL

FULL

SENSITIVE

INDICATOR

WEIGHT

RECOM-

ACID

ALKALINE

RANGE

pKf

MENDED*

COLOR

COLOR

pH

Meta-cresol purple

(acid range)

382.414

0.04

Red

Yellow

1.2-2.8

1.5

Thymol blue (acid

range)

466.570

0.04

Red

Y^ellow

1.2-2.8

1.5

Brom phenol blue

669.994

0.04

Yellow

Blue

3.0-4.6

4.1

Brom chlor phenol

blue

581.076

0.04

Yellow

Blue

3.0-4.6

4.0

Brom cresol green

698.046

0.04

Yellow

Blue

3.8-5.4

4.7

Chlor cresol green

520.210

0.04

Yellow

Blue

4.0-5.6

4.8

Methyl red

269.294

0.02

Red

Yellow

4.4-6.0

5.1

Chlor phenol red

423.260

0.04

Yellow

Red

4.8-6.4

6.0

Brom phenol red

512.178

0.04

Yellow

Red

5.2-6.8

6.2

Brom cresol purple

540.230

0.04

Yellow

Purple

5.2-6.8

6.3

Brom thymol blue

624.386

0.04

Yellow

Blue

6.0-7.6

7.0

Phenol red

354.362

0.02

Yellow

Red

6.8-8.4

7.9

Cresol red

382.414

0.02

Yellow

Red

7.2-8.8

8.3

Meta cresol purple

(alkaline range)

382.414

0.04

Yellow

Purple

7.4-9.0

8.3

Thymol blue (al-

kaline range)

466.570

0.04

Yellow

Blue

8.0-9.6

8.9

Cresolphthalein

346.364

0.04

Colorless

Red

8.2-9.8

9.4

Phenolphthalein

318.312

0.04

Colorless

Red

8.3-10.0

9.7

*In 95% ethyl alcohol.

f Approximate invert logarithm of apparent dissociation constant.

type of indicator the H-ion concentration may be determined by
the alteration in intensity of color, with the second type by altera-
tion in hue. The two-color indicators, such as the sulfonphthaleins,
are more satisfactory because one can measure changes in hue
more accurately than changes in color intensity, especially when
one must depend on rather crude methods of colorimetry as is
usually the case when indicators are employed.

The sulfonphthalein indicators are especially valuable as indi-
cators for still other reasons. Unlike the azo compounds, such as
methyl red, they are very stable chemically, while they are less

Xanihene Dyes 201

affected by the presence of neutral salts and proteins than are
many other indicators.

The first list of sulfonphthalein indicators was published by
Clark and Lubs (1917), who to make their series complete had to
include methyl red, an azo dye, less satisfactory because its solu-
tions are subject to reduction on standing. More recently Cohen
(1923, 1926) added six new sulfonphthalein indicators, among
them two (brom cresol green and chlor cresol green) which have
sensitive ranges so close to methyl red as to make the latter
unnecessary.

These indicators, unlike the usual dyes, are ordinarily furnished

in the form of color acids, which are practically insoluble in water.

They must accordingly be employed in alcoholic solution or must

be converted into the di-sodium salt with the proper amount of

NaOH, the quantity of the latter to use being figured by the

following formula:

TTT • 1 . ^- /-wTT Weight indicator X 40

\\ eight ^ aOH = ^ ■,.., • — 7—'

Molecular weight indicator

The alcoholic solutions are simpler to prepare and for ordinary
indicator purposes are equally satisfactory; they cannot, of course,
be employed, in vital staining.

Table 3, above, quoted from Leaflet IX of the Manual of
Methods for Pure Culture Study of Bacteria (Soc. of American
Bacteriologists; published by Biotech Publications, Geneva,
N. Y.), shows the color changes and sensitive ranges of the in-
dicators described below. (This table also includes methyl red,
which is an azo dye, and is described in Chapter V of this book.)

In the following list the compounds are arranged in the order
of their dissociation constants and hence in that of their sensitive
ranges in the pH-scale.

»5 META-CRESOL PURPLE

pK = 1.5, 8.3

This indicator is 7?i-cresol sulfonphthalein.

HO OH

/ C \
CH3 / -O CH3

so,

An indicator changing from red to yellow in its acid range and
from yellow to purple in its alkaline range.

202 Biological Stains

slO THYMOL BLUE

pK=1.5, 8.9
This is thymol sulf onphthalein :

CH3 CH3 CH3 CH3

CH CH

HO I I OH

\/\

CH3 c _ (:h3

/ o

so.

Thymol blue is now a well known indicator, used both in its acid
range (red to yellow) and in its alkaline range (yellow to blue).
In the latter range it may well be replaced with meta-cresol purple.

si 5 BROM PHENOL BLUE

pK = 4.0

This is tetra-bromo-phenol sulfonphthalein.

Br

OH

An indicator changing from yellow to blue.

«20 BROM CHLOR PHENOL BLUE

pK = 4.0
This is di-bromo-di-chloro-phenol sulfonphthalein.

CI

OH

An indicator almost identical with the last in range, and showing
the same change in color.

Xanthene Dyes 203

s25 BROM CRESOL GREEN

pK = 4.7

This is tetra-bromo-meta-cresol sulfonphthalein

Br

Br

ivj

yJt

/\/

/\/ Br
CH3

Br \/\

/ c

CH3 /

SO.
An indicator changing from yellow to blue.

s30 CHLOR CRESOL GREEN

pK = 4.8
This is tetra-chloro-meta-cresol sulfonphthalein.

CI CI

HO I I OH

ci-\/\ /\/-ci
/ c_ \

CHj / O CH3

I I SO,

An indicator changing from yellow to blue through a range only a
little more acid than that of methyl red.

sS5 CHLOR PHENOL RED

pK = 6.0

This is di-chloro-phenol sulfonphthalein.

HO OH

CI C_ CI

/ O

I ! so.
\/

An indicator changing from yellow to red.

204 Biological Stains

s40 BROM PHENOL RED

pK = 6.2

This is di-bromo-phenol sulfonphthalein.

HO OH

\/\ /\/

Br C_ Bi

/ O

1 I SO.

\/

An indicator changing from yellow to red through a range almost
identical with that of the better known brom cresol purple. It is
recommended in place of the latter because it is free from trouble-
some dichromatism.

45 BROM CRESOL PURPLE

pK = 6.3

This is di-bromo-ortho-cresol sulfonphthalein:

CH3 CH3
HO I I OH

Br C_ Br

O

s \

rsoa

A well-known indicator changing from yellow to purple. It has
been used by McMaster and Parsons (1938) as a vital stain.

850 BROM THYMOL BLUE

pK = 7.0
This is di-bromo-thymol sulfonphthalein:

OH3 v^H3 CH3 CH3

CH CH

HO I I OH

Br I \ / I Br
CHj C CH3

/ o

,/\ I

I SO.

Xanthene Dyes 205

It is a very valuable indicator because of its dissociation constant
at close to true neutrality. It is yellow in acid, green in neutral,
and blue in alkaline solutions. It has also been employed as a
vital dye by McMaster and Parsons (1938), and as a stain for
fungi in roots by Garrett (1937).

S55 PHENOL RED

pK = 7.9

This is phenol-sulfonphthaleih:

HO OH

O

-I
SO

2

An indicator changing from yellow to red, employed for some time
in physiological work, and well known before the paper by Clark
and Lubs above mentioned. The range of this indicator (approx.
pH 6.8-8.4) makes it quite valuable in much biological work; it is
quite commonly used in the study of kidney function.

S60 CRESOL RED

pK = 8.3

This is ortho-cresol-sulfonphthalein :

HO OH

\/\

CH

An indicator changing from yellow to red

S65 CRESOLPHTHALEIN

pK = 9.4

This indicator is closely related to phenolphthalein, having the
constitution :

HO OH

CH

206

Biological Stains

It is a newer indicator than phenolphthalein, and is generally
regarded as preferable when used in conjunction with the sulfon-
phthaleins, because its range does not overlap quite so far the
alkaline range of thymol blue. It is a one-color indicator and its
color change is the same as that of phenolphthalein.

S70 PHENOLPHTHALEIN

pK = 9.7

This indicator, of which the formula is given on p. 198, is quite
useful in the alkaline range centering around pH 9.7, with a color
change from red in alkaline solutions to colorless in acid ones. It
has the disadvantage of a one-color indicator discussed on p. 200.

5. ACRIDINE DYES

There is a small group of dyes derived from the compound
acridine ^x ^

1
H

./

which is closely related to xanthene. They are not xanthene dyes,
but are included in this chapter for convenience. There are a few
of interest to the biologist.

To

ACRIDINE YELLOW

H

I
H.2N_/\_N_/\_,NH2

CHa

CI

CH.

C. I. NO. 785

-c=^

I

H

C15H16N3CI; Mol. Wt. 273.759
[A basic dye; absorption maximum about 455 (Formdnek)]

This dye is rarely called for in microtechnic, but has been re-
ferred to by Metcalf and Patton (1944) as a fluorochrome useful in
insect histology.

Xanthene Dyes

207

TlO

CHj

CORIPHOSPHINE O

H

1
N /\

C. I. NO. 787

\ /'

N'

CHa

H

/

N— H

\
CI

CH3

Tl5

-c="

I

H

CieHisNsCl; Mol. Wt. 287.785

[A basic dye; absorption maxima about 64-3, 4-82 {Formdnek)]

ACRIDINE ORANGE NO C. I. NO. 788

Synonyms: Basic orange 3RN. Euchrysine 3RXA.

CHg

CH3

H

N

CH2

/

CI

C^

I

H

C17H20N3CI; Mol. Wt. 301.811

[A basic dye, ordinarily obtained as the zinc chloride double salt;
absorption maxima about {467) 4-97 (Formdnek)]

Both of these dyes are among those employed as fluorochromes
by Metcalf and Patton (1944). Strugger (1948) has used acridine
orange for demonstrating bacteria in soil suspensions, by means
of ultraviolet light; bacteria are green, humus particles red. Their
chief use, in fact, is as fluorochromes; but acridine orange has re-
cently attracted some attention because it stains tumor cells
selectively, intra vitam, and causes retardation of tumor growth
(Lewis and Goland, 1948).

t20

H5C2— O

RIVANOL

H

^N— O— CO CHOHCH3
H

H

I

N

~C=

1
NH2

C18H21N3O4; Mol. Wt. 343.372

This has also been called for by the two authors above men-
tioned, as a fluorochrome.

208

Biological Stains

T25 ACRIFLAVINE C. I. NO. 790

Synonym: Trypaflavine. Flavine.

H H
CH3 CI I /

11— N \ / N— H

H I J 1 f CI

\/~C=\/

H

C14H15N3CI2; Mol. Wt. 296.198
{A basic dye)

This yellow dye is one of those developed by Ehrlich for its
therapeutic value. It is marketed at present as a disinfectant.
It is employed as a bacteriostatic agent by Churchman (1927)
mixed with methyl and crystal violets under the name of "acri-
violet." Acriflavine has little use as an ordinary stain, but is
coming to be employed to some extent as a fluorochrome; see
Levaditti et al (1940), Metcalf and Patton (1944).

t30

NEUTRAL ACRIFLAVINE

Synonyms: Neutral trypaflavine. Eafl^vine.

Gonacrin.

NH.

CHj CI

NH,

Neutroflavine.

C
H

C14H14N3CI; Mol. Wt. 259.733

This dye finds use as a disinfectant. It is said to be less irritat-
ing than acriflavine, because it is neutral.

t35

H2N

PROFLAVINE

\ _N^/\_NHo-H.2S04

C. I. NO. 790

H

C13H12N3SO4; Mol. Wt. 306.310
(A basic dye ordinarily supplied as the sulfate)

Like the very similar acriflavine, this is used primarily as a
disinfectant. It has been called for by Rogers (1940) as an agent
for inhibiting sulfate-reducing bacteria.

Xanthene Dyes 209

t40 ATABRINE :- Q -l.«- vu»^C/i'*'*^j|^

Synonym: Atehrine (in Germany).

NH-CH(CH3)CHoCH2CH2N(C2H5)2

CH30_/X_C_.

I I

C23H31N3O; Mol. Wt. 365.502

This is a yellow compound used primarily for its medicinal
value (as an antimalarial agent). It shows fluorescence in ultra-
violet light, and has been called for by Metcalf and Patton (1944)
as a fluorochrome in insect histology.

Atebrine and a few other acridine antimalarials are among the
dyes which Lewis and Goland (1948) have found to stain and to
retard tumors in mice.

t45 PHOSPHINE C. I, NO. 793

Synonyms: Leather yellow. Xanthin.

A mixture of nitrates of chrysanilin, and probably some of its
higher homologs, chrysanilin having the following formula:

H NO3

\

C19H10N2O2; Mol. Wt. 348.350
{A basic dye; principal absorption in the neighborhood of

This dye has been used (in 1% solution) by Schumacher (1922)
following methylene blue (1% phenolic solution) to stain bacterial
nucleins and nucleoproteids yellow while the free or unbound
nucleic acid is green. Metcalf and Patton (1944) employ phosphine
3R, one of the redder shades of this dye, as a fluorochrome in the
study of insect histology by fluorescence microscopy.

210 Biological Stains

t50 RHEONINE a C. I. NO. 795

Synonyms: Rheonine ALy G or N. Fast phosphine NAL.

CH3

H

\

N

_/\-

N /\-

-NH2

CHa

\y-

\

\
CI

1 \

CH3

\

N/
CH3

{A basic dye; absorption maximum about ^6^.5)

Although seldom called for in biological work, this dye is men-
tioned by Pick (1935) as a fluorochrome useful in vital staining of
the frog.

CHAPTER IX
NATURAL DYES
THE NATURAL DYES

AS STATED in Chapter II the group of natural dyes is
l\ shrinking, as more and more of them are being produced by
■^ -^ artificial means. Alizarin, for example, in the form of
madder, used to be extracted from the roots of Ruhia tinctor-
um; but the artificial manufacture of 4:his dye is now much more
economical. The group of natural dyes, as ordinarily recognized,
contains only those which are not yet produced by artificial
means. Indigo, however, is listed in this chapter, because in its
chemistry it does not fall in well with any of the groups of artificial
dyes. It is still obtained in part, moreover, from the indigo plant,
although under present-day conditions its artificial manufacture
is ordinarily the more economical. Similarly, orcein can now be
synthetically prepared, but is still included in this chapter because
of its relationship to other dyes that are secured from natural
sources only.

The chemistry of the natural dyes is less definitely known than
that of the artificial dyes. This is easily understood; for it will be
recalled that there are two ways of obtaining information as to the
chemistry of unknown compounds: the first by decomposing them
into simpler compounds of known composition; and the second by
manufacturing them from known compounds. In the case of dyes
not yet prepared artificially the second of these two lines of pro-
cedure is out of the question; hence there is special difficulty in
learning their exact chemical structure.

The most important natural dyes for the biologist are hema-
toxylin, indigo, cochineal (and its derivatives), orcein, and litmus.

u5 Saffron

A natural pigment of uncertain composition extracted from the
stigmas of Crocus sativus. It has been employed by Masson (see
Foot, 1933) in a connective tissue stain. A special application of
this procedure has been made by Block and Godin (1936) for
staining yellow fever lesions in the liver. A later application by
Van Hoecke and Sebruyns (1952) calls for this dye, after Bismarck
brown, aniline blue W.S. and hematein, as a differential stain for
glandular cells of the stomach.

The Indigo Group
uio INDIGO c. I. NO. 1177

Synonym: Indigo blue.
The plants from which indigo was formerly exclusively manu-

211

212 Biological Stains

factured are largely species of the genus known as Indigofera, al-
though some indigo-bearing plants are recognized by botanists as
belonging to different genera. In these plants is a glucoside,
indican, which is converted by fermentation into the dye indigo.
Various formulae have been given for indigo; the one favored at
present is based upon its method of artificial manufacture:

rY

CO CO

c=c I I

\ / \ /\/

NH NH

C16H10N2O2; Mol. Wt. 262.256

In this formula the exact chromophore group is uncertain; but
the ketone group (CO) in a closed ring occurs so often in dyes that
it is regarded as probably having chromophoric properties; the
same is true of the C = C linkage.

ul2 INDIGO-CARMINE C. I. NO. I180

Synonym: Indigotine la.
This is the sodium salt of indigo disulfonic acid:
NaS03 CO CO SOsNa

Ci6H8N208S2Na2; Mol Wt. 466.354

Indigo carmine is a blue dye of acid properties, which is sometimes
used as a plasma stain in contrast to some red dye such as carmine
or basic fuchsin (see Shumway, 1926), sometimes hematoxylin.
A few special applications of the stain can be mentioned: by
Petragnani (1928), mixed with acid fuchsin, as a stain for Negri
bodies; by Cuyler (1932), mixed with eosin Y, as a counterstain to
hematoxylin for staining vaginal smears; by Brozek (see Hruby,
1933) in plant cytology where it is used in picric acid solution in
contrast to basic fuchsin; by Kempton, Bott and Richards (1937)
for uretral injection in studies of glomerular excretion.

For use in Fraenkel's method for elastic tissue, see Staining Procedures, p.IBs-lT.
ul5 BeRBERINE C. I. NO. 1137

Berberine was originally obtained from barberry and is some-
times called barberry extract. It is found in other plants, how-
ever, and can also be prepared synthetically. It has the following
formula:

Natural Dyes 213

O— CH.

I I
/\_0
H I I

I

I CH2

CH3 0_| I N_ I

\/\ / CU,
CH3O/ c

l\

H OH
C20H15NO5; Mol. Wt. 353.360

This compound acts as a basic dye. It has rarely been specified
in biological work; but Metcalf and Patton (1944) mention it as
a fluorochrome in connection with insect histology.

U20 Cochineal Products c. i. no. 1139

Cochineal is a dye that has long been well known. It is obtamed
from a tropical insect generally known as the cochineal insect. By
grinding and extracting the dried bodies of the female of the species
in question a deep red dye is obtained, which is known as cochineal.
On treatment with alum this solution yields a product somewhat
more free from extraneous matter, known as carmine. This is the
form in which the dye is generally obtained by the microscopist.
Cochineal products are used in various ways in microscopic technic,
generally as nuclear dyes. They are extremely valuable in cases
where it is desirable to stain in bulk before sectioning.

Cochineal, itself, has been used for various purposes in micro-
scopic technic, even though less used today than carmine. Alone it
has little value, to be sure, for it has no direct affinity for tissues un-
less they contain iron, aluminium or some other metal. It is most
commonly employed either with or following a salt of one of these
metals as a mordant. A tincture of cochineal, that is an alcoholic
solution containing calcium and aluminium chlorides, has been
used by Mayer (see Lee, 1937, p. 149) both on sections and for
staining in bulk; but its most common method of use is with alum
in watery solution. An alum-cochineal of this sort was first used
independently by Mayer (1878, 1892) and Czokor (1880); it can
be used for sections, and is specially recommended for staining in
bulk, by which technic it stains nuclei violet red, and blood and
muscle cells orange, while the cytoplasm is but weakly colored.
A chrom-alum-cochineal has been used by Hansen (1905) for stain-
ing sections. Spuler (1901) recommends an iron-alum-cochineal
for staining in bulk when the sections are to be photographed, the
technic bringing out nuclei, the blood in the tissues, and the muscle
striations; sections may also be stained by the same method. By

214 Biological Stains

this techiiic the iron alum is applied first to the tissues as a mor-
dant, and then followed by the stain. In Hansen's ferri-cochineal,
on the other hand, the iron alum is mixed with the dye, and the
mixture used for staining sections of tissue (see Lee, 1937, p. 145).
Reynolds (1936) has proposed a mixture of alum cochineal with
hematoxylin in staining trematodes and nematodes, in toto.

Carmine. Carmine is of considerable historic interest. It was
used as early as 1770 by Hill and in 1838 by Ehrenberg, although as
stated elsewhere not exactly for histological purposes. It was
also employed in 1849 by Goppert and Cohn, by Corti in 1851,
and by Hartig in 1854-8, these being the first uses of dyes in
histology. It is still a valuable stain today, in spite of the enor-
mous variety of synthetic dyes now available. It is much used for
staining in bulk, particularly in embryological work. A well-
known formula is Schneider's aceto-carmine (see Lee, 1937, p,
142), which is a valuable chromatin stain for fresh material in
smear preparations. Belling's (1921) iron aceto-carmine is
valuable for staining chromosomes in smear preparations from
anthers. Alum carmine was used by Grenacher (1879) for similar
purposes. Carmine is only slightly soluble in water at a neutral
reaction; so solutions must be either acid (like the three above) or
alkaline. Three alkaline formulae are of considerable use: am-
monia carmine, which has been used both for injection and
for staining sections; soda carmine, used primarily for injection;
and Mayer's (1892, 1896) magnesia carmine, useful either for sec-
tions or for staining in bulk. Orth (1883) proposed a lithium
carmine (i.e., dissolved in a solution of Li2C03) as a nuclear stain
for tissues; and the formula for this stain, as given by Krause
(1926, I, p. 265), is still frequently employed. Alcoholic solutions
are also used : Grenacher's borax carmine (or as modified by Mayer,
1892, 1896) being a splendid nuclear stain for sections; and the
hydrochloric carmine of Mayer serving both for sections and for
staining in bulk. A special formula containing aluminium chloride
(known as muci-carmine) has been proposed by Mayer (1892,
1896) and is used for staining mucin. In double staining it is
sometimes used with indigo carmine; but most often with picric
acid or spirit blue. Picro-carmine is a very well-known combina-
tion used for double staining effects in sections, particularly for
nervous tissue; it stains nuclei red and cytoplasm yellow.

One of the important uses of carmine is in Best's (1906) carmine
stain for glycogen. The method is simple and the result beautiful,
the red glycogen standing out in sharp contrast to the blue of the
nuclei after staining in alum hematoxylin. The stain is perma-
nent; the method is of much importance both to the pathologist
and to the histologist.

Carminic acid. The dye principle of carmine and cochineal is
carminic acid. This product is obtained by extracting the insect

Natural Dyes 215

bodies with boiling water, treating the extract with lead acetate or
barium hydrate, and then decomposing the lead or barium carmin-
ate with sulfuric acid. The exact composition of carminic acid is
still somewhat uncertain; so far as known, it is:

CH3 OH

CO I CeHiiOs

HO / CO I OH
COOH OH

It is a fairly strong dibasic acid and forms readily soluble salts with
the alkali metals, and insoluble salts with the heavy metals.
Aluminium carminate (obtained by precipitation from aluminium
acetate and carminic acid or ammonium carminate) is soluble in
aqueous or weak alcoholic solutions of acids.

A slightly different aluminium compound, formed by mixing
alum and carminic acid is used in histology. This combination
was called carmalum by Mayer (1892, 1899), and has also been
used by Grenacher (1879) and Rawitz (1899); it is a useful nuclear
stain for sections; and is often employed with light green or indigo
carmine as a contrast stain. A so-called muci-carmine, an acid
solution containing aluminium chloride, has been employed by
Rawitz to stain mucin; while Mayer's para-carmine, containing
aluminium and calcium chlorides, is used both for sections and for
staining in bulk. By others a combination of iron with carminic
acid has been used for similar purposes.

Carmein. Carmine, kept in ammoniacal solution, changes in its
properties, due to oxidation. The oxidized carmine, often known
as carmein, can be obtained by treating a carmine solution with
hydrogen peroxide and precipitating with alcohol. It is a dark
colored mass which can be ground into a black powder.

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH
COCHINEAL DERIVATIVES ARE USED.*

NAME OF PROCEDURE

PAGE REFERENCE TO

Staining Procedures

Mayer's mucicarmine stain

IA4-7

Mucicarmine with hematoxylin and metanil yellow

IA4-7

As nuclear stain with Weigert's resorcin fuchsin for elastin

IB3-I3

Best's method for glycogen

ID3-2I

Grenacher's borax carmine in plant histology

IIA-5

Aceto-carmine and basic fuchsin for pollen tubes in style

IIA-13

Belling's iron-aceto-carmine for plant cytology

IIB-3

Aceto-carmine with chlorazol black E in plant cytology

IIB-4

*Under this heading are given references to procedures described in detail in
Staining Procedures, edited by Conn and Darrow (1943-4).

216 Biological Stains

Orcein and Litmus c. i. no. 12.42

Both orcein and litmus are obtained from certain lichens, Lecan-
ora tinctoria and Rocella tinctoria. These lichens are colorless, but
when treated with ammonia and exposed to the air, blue or violet
colors develop. The colors are due to certain phenolic com-
pounds, one of which is orcinol:

OH

_/
CH3_/ \

\
OH

Orcinol, acted upon by air and ammonia, becomes orcein.

u30 ORCEIN

The exact formula of orcein is unknown. It is a weak acid,
soluble in alkalies, with a violet color. It is possible to synthesize
this dye from orcinol by the above-indicated process. As a
matter of fact very good synthetic products, for some purposes
better than the natural product, are now on the market. The
synthetic orceins from different manufacturers, however, are pre-
pared by different methods and are not the same in their behavior.
The reason for these differences are still unknown. The method
of manufacture has never yielded exact information as to its struc-
tural formula; but it is believed to contain in its molecule the resi-
dues of four orcinol molecules. It has some indicator properties
both for pH and oxidation-reduction; but is not as useful in that
respect as its closely related litmus. It is a weak acid, violet in
alkaline solution; in acid solutions it is orange-red.

Unna (see Lee, 1937, p. 451) used orcein in alcoholic solution for
staining elastin tissue; he employed it for connective tissue, follow-
ing polychrome methylene blue; and for plasma fibrils in the
epithelium, following anilin blue; also with anilin blue or acid fuch-
sin in studying the process known by him as chromolysis. It has
found less frequent use among other histologists; but has been
employed in acetic acid solution for staining sections or dissolved
in weak hydrochloric acid, for staining sections of embryos; also
in combination with various other dyes for bulk staining or as a
connective tissue stain. Mollier (1938) calls for it in a quadruple
tissue stain, with iron-hematoxylin, naphthol green B and azo-
carmine G. Kornhauser (1943) uses it in a somewhat different
quadruple tissue stain with acid alizarin blue, orange G, and fast
green FCF. LaCour (1941) employs it in a stain-fixative for
cytological work, either for plants or animals. For this last men-
tioned purpose the synthetic orcein has not worked as well as that

Natural Dyes 217

derived from natural sources; but as an elastin stain, and in the
Mollier and Kornhauser quadruple stains, the synthetic orcein
seems to be preferable.

For the technic of the MoIIIer quadruple stain, see Staining Procedures, p. IB3-7.

For the technic of the Kornhauser quadruple stain, see Id., p. IB3-8.

For the technic of the Tanzer-Unna orcein stain, see Id. p. IB3-I6,

For Fraenkel's method for elastic tissue, see Staining Procedures p, IB3-I7.

U35 LITMUS

The exact composition of litmus is likewise unknown. It is ob-
tained from the same lichens as orcein, treating them with lime
and potash or soda, in addition to air and ammonia. Its primary
colored principle is known as azolitmin; but there must be other
colored compounds in litmus, because the color reactions of azo-
litmin are not exactly the same as those of litmus. Azolitmin
may or may not be a single chemical compound ; and in any case, its
chemical formula is unknown. It can be synthesized from
orcinol by methods very similar to those used in preparing orcein,
and the synthetic product is very much like the azolitmin obtained
from the lichens, if not identical with it. The color reactions of
either litmus or of azolitmin are very similar to those of the in-
dophenols (see p. 102); but it is difficult to figure how an indo-
phenol could be derived from orcinol by the process employed.

Litmus is a feeble dye and is never used as an histological stain.
Its classic use is for indicator purposes; at one time it was the best
acid-base indicator known. Since the importance of learning
actual pH of solutions came to be realized, however, litmus has
proved too inaccurate an indicator, and has largely been replaced
for chemical use by synthetic dyes (notably the sulfonphthaleins)
which change color through a pH-range near the neutral point
and which are not subject to loss of color on reduction.

This loss of color by reduction, which is an inconvenience to the
chemist who wishes to use litmus as a pH indicator, happens to be
of advantage to the bacteriologist. Litmus has been employed
for over 50 years as an indicator in bacteriological media; and in
some of these media (especially milk) it serves to indicate changes
in pH and in 0-R potential simultaneously. Although chemi-
cally speaking, it is a decidedly inaccurate indicator for either
purpose, it has served the bacteriologist to bring out differences be-
tween species in a way that no other indicator alone has proved
able to do. Azolitmin, either the synthetic product or that de-
rived from the lichens, does not serve the purpose, chiefly because
it does not have exactly the same sensitive range in relation to the
pH-scale as does litmus.

It is of interest to add that chemists have been telling bacteriol-
ogists for years that they should discard litmus in favor of more ac-
curate indicators, using one for pH, another for 0-R potential.

218 Biological Stains

It required the second World War, however, with its interrup-
tion of trade and resulting cutting off of the supply of litmus, to
stimulate bacteriologists to investigate combinations of indicators
to replace litmus. Ulrich (1944) proposed a mixture of methylene
blue (to indicate reduction) with chlor phenol red (for pH) which
seemed, for some kinds of bacteria at least, to be preferable to
litmus as an indicator in milk. This combination, however,
shows no sign of replacing litmus for the purpose.

Brazilin and Hematoxylin

The two natural dyes, hematoxylin and brazilin, are closely
related chemically and upon decomposition yield the two com-
pounds, pyrocatechol

OH

OH
and pyrogallol

OH

I OH

OH

Both dyes are obtained by extraction of the bark of certain trees,
hematoxylin from logwood and brazilin from brazil wood (red
wood). Both trees are legumes and belong to the family Cesal-
piniaceae; they are found only in the tropics. Hematoxylin comes
from a single species ; while brazil wood is a term applied to various
different species all yielding brazilin.

U40 BRAZILIN C. I. NO. 1143

The composition of this substance is supposed to be:

HO /\ O CH2

C— OH

CH \
CH2

HO OH

Natural Dyes 219

Its solution is colorless, but it becomes red on exposure to the air,
as it is then oxidized into the dye brazilein, which probably has the
formula :

HO /\ O CH

C— OH

\
_CH2

HO O

With alum it is employed as a nuclear stain (known as brazalum)
by Mayer (see Lee and Mayer, 1907, p. 218). It is also used by
Hickson (1901) for similar purposes following treatment with iron
alum as a mordant. Belling (1928) has employed it in place of
carmine for staining plant chromosomes in fixed or fresh material,
and Shaffer (1933) for distinguishing paper fibers bleached with
sulfite from those bleached with sulfate.

U50 HEMATOXYLIN C. I. NO. 1146

Hematoxylin, as is well known, is a constituent of logwood, a
product of South America. It was first obtained about 1840 by
extracting logwood chips with ether, evaporating, digesting with
alcohol and standing with water after distilling off the alcohol;
hematoxylin was found to separate out in crystals. A little later
a similar method was employed, but instead of using logwood
chips, the starting point of the process was with commercial log-
wood extract, which is the dried aqueous extract of the wood.
In one modern method of manufacture the dried commercial ex-
tract is extracted with ether in a continuous extraction apparatus,
evaporated to dryness, dissolved in water, filtered and crystallized
out of the solution. All of these steps, particularly the ether
extraction, are slow and difficult to handle on a factory scale,
requiring special expensive apparatus.

The American manufacture of hematoxylin started during the
first World War, just as in the case of the synthetic dyes. The
problems presented by its manufacture, however, were quite differ-
ent from those presented by the synthetics. They were discussed
at about that period by McClung (1923) and Conn (1927). Briefly
it can be explained here that the first American hematoxylins,
marketed during that war, were very crude and proved quite un-
satisfactory; but that during the decade following the war, fre-
quent improvement in the supply was made by the manufacturers.
The first American product was dark in color; and there was so
much demand for a light colored hematoxylin that the manu-

220

Biological Stains

facturers introduced the process of bleaching with sulfur dioxide.
It was subsequently found that this bleaching injured the staining
qualities, and the manufacturers changed their method and began
turning out a darker colored but more satisfactory stain. About
1926 another change in process of manufacture was made and the
staining properties seemed thereby to be improved. Until re-
cently there has been only one American manufacturer of hema-
toxylin whose product could qualify as a biological stain; but the
great demand caused by the second World War has resulted in
other companies entering the field and developing cheaper pro-
cesses of manufacture. These cheaper methods of production
yield a darker colored and presumably less pure product; it is not,
however, to be compared with the crude material put on the
American market during the first World War, as it seems to be a
satisfactory stain in spite of impurities that may be present.

Hematoxylin is similar to brazilin, but has one more hydroxyl
group, the generally accepted formula being:

OH

HO

O CH

CH

C— OH

■ \
CH2

HO HO

Like brazilin, it is not a dye, but its color develops in solution upon
standing, due to the oxidation into hematein, which is homologous
to brazilein and probably has the formula:

OH

HO

.0_CH2

C

C— OH

X
CH,

HO O

Hematein is available commercially in dry form. Less atten-
tion has been given to its production than to that of hematoxylin.
Hence it frequently results that a hematein sold by some company
is more crude than their hematoxylin, although theoretically
hematein is a derivative of hematoxylin.

Natural Dyes 221

Hematoxylin is without question one of the most important
biological stains. It is as valuable to the cytologist and histologist
as methylene blue is to the bacteriologist; and probably is second
only to methylene blue in the number of different purposes for
which it is used. It is valuable not only because it is a powerful
nuclear stain and a chromatin stain par excellence, but also be-
cause it has striking polychrome properties. With the proper
differentiation it is possible to get several shades intermediate
between blue and red to show in the same preparation.

Hematoxylin is seldom used alone, as it has little affinity for the
tissues in itself, even after "ripening" when it is largely converted
into hematein. Some form of mordanting is ordinarily required;
and most of the hematoxylin formulae either call for some metallic
salt or specify previous treatment of the sections with one. In
plant histology, however, there is some use for hematoxylin alone.
Its greater affinity for plant than for animal tissue implies the
presence of aluminium, copper, or iron in the former. In fact
hematoxylin can be used as a very delicate reagent for iron or
copper.

Perhaps the best known formulae for staining with hematoxy-
lin are the combinations with aluminium, generally in the form oi
alum. Bohmer's alum hematoxylin (1865), although no longer
employed, is of historic interest as it was the first stain of this type
to be used. The best known at present is Delafield's alum hema-
toxylin (see.Prudden, 1885), which is a very useful tissue stain
with great affinity for chromatin and nuclei, and has much value in
staining cellulose walls in vascular plants. Another alum hema-
toxylin used for similar purposes is that of Ehrlich (see Krause,
1926-7, p. 972).

Mayer's hemalum (1891, 1899) is another well-known alum
combination. In this stain hematein is first prepared and then
combined with alum. The name hemalum, proposed by Mayer, is
now generally accepted for this combination, and various other
hemalum formulae have since been proposed. They are useful
chromatin stains and are called for in various special procedures.

Mayer (1891, 1899) also combined hematein with aluminium
chloride, his hemacalcium calling for this salt and calcium chloride,
while his muci-hematein contains aluminium chloride and glycerin.
The latter is used for staining mucin.

The iron combinations are perhaps equally valuable. The
original iron hematoxylin was that of Benda (1886); but the best
known at present is M. Heidenhain's (1892, 1896), which is one of
the most useful histological and cytological stains, both in botany
and zoology. It is a powerful stain for chromosomes and centro-
somes, and is of use for bringing out the middle lamellae in wood.*
Various other modifications of iron hematoxylin have been used,
but they are all similar in principle. Ordinarily the iron salt is

222 Biological Stains

not mixed with the stain, but is used for a preHminary mordanting
of the tissue.

Hematoxyhn has been combined with chromium, one of the
early staining methods. being that of R. Heidenhain (1886), which
called for potassium bichromate as a mordant. Various recent
modifications are in use today, such as that of Apathy (1888), for
staining general tissue. Weigert (1884) used a chrom combina-
tion for staining nervous tissue.

Benda (1893) employed hematoxylin following treatment with
a copper salt for studying spermatogenesis; and Bensley (see
Guyer, 1936, p. 164) a similar technic for chromosomes and mito-
chondria. A formula containing logwood extract, with alum and
copper sulfate was suggested by Cook (1879).

Mallory (1938, p. 156) proposed a formula for hematoxylin con-
taining phosphomolybdic acid and also one containing phospho-
tungstic acid. The latter method is especially valuable for stain-
ing cells in the process of mitosis, and for distinguishing fibroglia,
myoglia and neuroglia fibrils from collagen and elastin fibrils,
especially in tumors, but also in normal tissues. It brings out
sharply the striations in skeletal and cardiac muscle fibers. Hema-
toxylin is used in combination with other stains, especially eosin,
but not so frequently as in the case of the common anilin dyes.
The Van Gieson technic calls for hematoxylin followed by picric
acid and acid fuchsin. A few other methods call for picric acid or
ammonium picrate after hematoxylin: and it is sometimes used
with eosin or after orange G or acid fuchsin. Most of these com-
binations, however, are called for only in the case of special pro-
cedures.

Isohematein. If hematein is treated with HCl in sealed tubes
at 100°C and then treated with silver hydroxide to remove the
chlorine, a solution is obtained of a compound known as isohem-
atein, which can be secured as an amorphous mass by evaporation.
At the request of the Stain Commission, an American manu-
facturer of logwood products actually prepared an experimental
batch of isohematein by this method, and Cole (1931) reported on
its use as a biological stain. The result of this study was to in-
dicate that it could hardly replace hematoxylin as a routine stain,
but that on account of its higher tinctorial power it may have
value for some special purposes such as the selective staining of
nerve cell bodies of fibrillae and cross striations in muscle cells. It
has never been made available commercially, and at present there
does not seem to be suflBcient demand for it to justify its production.

Natural Dyes

223

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH
HEMATOXYLIN AND HEMATEIN ARE USED

NAME OF PROCEDURE

PAGE REFERENCE TO

Staining Procedures

Mayer's mucihematein, Laskey modification

IA4-8

Delafield's hematoxylin in animal histology

IA4-II

Heidenhain's hematoxylin in animal histology

IA4-I2

Mayer's hemalum and eosin bluish

IA4-I2

Mayer's hemalum with Congo red

IA4-I3

Mayer's acid hemalum, Hllie modification

IA4-I3

Mallory's phosphotungstic acid hematoxylin

IA4-I4

Weigert's iron hematoxylin with safranin

IA4-6

Hematoxylin with Van Gieson stain

IA4-2I

Grossman modification of Mallory'sjconnective tissue stain

IB3-5

Hematoxylin with Biebrich scarlet and picro aniline blue

IB3-I2

Mucicarmine with hematoxylin and metanih yellow

IA4-7

Verhoeff's connective tissue stain

IB3-I4

Modified Gallego elastic tissue stain

IB3-I5

For nuclei in Tanzer-Unna orcein method

IB3-I6

Masson's trichrome stain

IB3-I8

Bielschowsky's connective tissue stain. Foot's modification

IB3-2O

Same; Wilder's modification

IB3-22

Pal-Weigert method for myelin sheaths

IC3-28

Hematoxylin with Herxheimer's Sudan IV fat stain

ID3-I8

Lorrain-Smith-Dietrich method for lipoids

ID3-I9

Proescher's oil-red-t)yridine for lipoids

Best's method for glycogen

Bauer's leucofuchsin method for glycogen

Heidenhain's hematoxylin for plant histology

Delafield's hematoxylin for plant histology

Iron alum hematoxylin and safranin for plant histology

Hematoxylin for permanent smear preparations of sporo-

cytes
Heidenhain's hematoxylin in plant cytology
Regaud's iron alum hematoxylin in plant cytology
Gram-Weigert technic for bacteria in tissues
Weigert's stain for fibrin and bacteria in tissues
With Verhoeff's carbol fuchsin for tubercle bacilli in tissue

ID3-I9
ID3-2I
ID3-22

IIA-7
IA-8

IIA-9

IIB-6

IIB-8

IIB-9

IIIB3-8

IIIB3-9

IIIB3-IO

CHAPTER X

MISCELLANEOUS DYES, PIGMENTS, AND HISTO-

CHEMICAL REAGENTS

1. THE ANTHRAQUINONE GROUP
The anthraquinone dyes include derivatives of anthracene,

224 Biological Stains

through its oxidation product anthraquinone:

O

These dyes show the quinoid ring, which is the most important
chromophore in nearly all the dyes to be discussed in the three fol-
lowing chapters; it forms very strong chromogens, which require
only the addition of auxochrome groups to be converted into strong
dyes, either basic or acid. The chromogen anthraquinone is con-
verted into a dye by the addition of hydroxyl groups, its best
known. derivatives among the dyes being: 1:2 dihydroxy-anthra-
quinone (alizarin) and 1:2:4 trihydroxy-anthraquinone (purpurin).
Both of these compounds occur in nature in the root of madder,
being the colored principles of madder extract. They have the
property of combining with metallic oxides to form so-called
"lakes," insoluble compounds of different color from the dye enter-
ing into them. This makes them valuable ones to use after mor-
danting with aluminium, iron or chromium compounds.

El

ALIZARIN C. I. NO. IO2.7

(Various grades denoted as: Alizarin P, VI, le.)

C14H8O4; Mol. Wt. 240.204
(An axdd dye; absorption maxima about \610.8], 566.5^ \527.6\

in alkaline solution)

Solubility at 26°C: in water nil; in alcohol 0.125%

225

226

Biological Stains

^ Alizarin stains tissues a feeble yellowish red if used on them
directly. In the presence of aluminium compounds intense red
colors are formed ; bluish violet in the presence of iron ; and brown-
ish violet in the presence of chromium. It has been used as a stain
for nervous tissue and for the vital staining of protozoa. Geyer
(1932) employs it to distinguish cows' from goats* milk. The chief
present use of alizarin, however, is as an indicator.

H r
N

I

I

I

I

o

I

O400
o

I I I • I I I I I I X • I J
460 520 580 640 700
WAVELENGTH m }X

Fig. 26. Spectral curve of alizarin red S.
b5 alizarin red S C. I. NO. IO34

Synonyms: Alizarin red, water soluble. Alizarin carmine.

SOjNa

CuHyOxSNa; Mol. Wt. 342.253
{An acid dye)
{Absorption maxima in alkaline solution about 557 [59J!t.-9])

Solubility at S6°C: in water 7.69%; in alcohol 0.15%
A spectral curve of this dye is given in Fig. 26.

Miscellaneous Dyes 227

This dye, sodium alizarin sulfonate, was used by Ben,da for stain-
ing chromatin in combination with crystal violet, the chromatin
staining brown, while the mitochondria stain violet. It has also
been used as a vital stain for nervous tissue in small invertebrates,
and by Schrotter (See Lee, p. 465) for sections of nerve tissue. A
recent application is for the gross staining of skeletons (Dawson,
1926, Lipman, 1935, Hollister, 1934); in fact, its most frequent
modern use is as a stain for bone. Backman (1935) employs it as
a chromosome stain in plant cytology.

For various methods calling for this dye in staining bone, see Staining Procedures,
pp. ID3-I2-I7.

ElO PURPURIN C. I. NO. 1037

Synonyms: Alizarin N0.6. Alizarin purpurin.

r

O OH

/ OH

II OH

O

CuHsOs; Mol. Wt. 256.204

{An acid dye; absorption maxima about [521.1], Jf85.5,

[It35.5] in alcohol.)

Purpurin is very similar to alizarin, but forms scarlet red lakes
with alumina. It has been used as a nuclear stain for histological
material, and for determining the presence of insoluble calcium
salts in the cell contents.

Ell QUINALIZARIN C. I. NO. IO45

Synonym: Alizarin Bordeaux BA.

HO O OH

II /

OH

y

HO
CuHgOe; Mol Wt. 272.204
{An add dye)

228 Biological Stains

This compound is seldom called for in biological work but has
been used by Broda (1939), mixed with azo blue and Titan yellow,
as an histochemical reagent for magnesium.

El3 CARYCINEL RED

O

_NH.C5Hu

O

C19H19NO2; Mol. Wt. 293.350
El 4 COCCINEL RED

O

II _NH-C5Hu

CsHn.NH O

C24H30N2O2; Mol. Wt. 378.496

These are two oil-soluble dyes first described and named by
Lillie (1945b). He regarded them, together with the following
dye (Oil blue NA) as good fat stains when employed in the super-
saturated isopropanol technic (Lillie, 1945 a). Coccinel red stains
fat scarlet, carycinel red a deep crimson.

El5 OIL BLUE NA

Probably of the following chemical formula:

O NHCbHu

O NH.CsHii

C24H30N2O2; Mol. Wt. 378.496
{Absorption maxima about \595], 64-0.)

This dye has come into prominence since 1940, as a stain for
rubber in plant sections. Whittenberger (1944) thus employs it
without a counterstain, while Addicott (1944) suggests either
safranin or Congo red for counterstaining. According to Lillie
(1945 a) it may also be used for staining fat in animal tissue.

For use in staining fat in tissue, see Staining Procedures, p. ID3-2O.

Miscellaneous Dyes 229

E20 ACID ALIZARIN BLUE GR C. I. NO. IO48

NH, O OH

HO I J I I SOjNu

NaS03/\/\/\/\0H

HO O NH.

CuHgNzOi 282X32; Mol. Wt. 506.334
(An acid dye)

Some acid alizarin blue, probably either this or the following,
has been employed by Buzaglo (1934) in combination with orcein
and gallocyanin as a tissue stain. Peterson (1924) has recom-
mended acid alizarin blue in a modification of the Mallory con-
nective tissue stain; the procedure seems to be well worth recom-
mending, but there proves to be some difficulty in standardizing
this dye for the purpose.

For the acid alizarin blue modification of the Mallory technic, see Staining Pro-
cedures, p. IB3-6, 8.

E«5 ACID ALIZARIN BLUE BB C. I. NO. I063

Synonyms: Anthracene blue SIVX, SWX extra

HO O OH

NaSO, I It I OH

HO 1 II I S03Na
HO O OH

CuHeOi 482X32; Mol. Wt. 508.302
(An acid dye)

This dye has come into use in connection with fluorescence
microscopy, because of the color which it imparts to various tissue
elements under ultra-violet illumination. Some anthracene blue
has been called for occasionally in aqueous solution with aluminium
sulfate. There are, however, two or three other anthracene blues
besides this dye; so it is not certain whether this is the one called
for in the technic in question.

e30 ALKANET

A natural dye of the anthraquinone group, closely related to
alizarin. It has rarely been specified for biological use, but Artsch-
wager (1943) has employed it as a stain for rubber in plant sections.

230 Biological Stains

2. THIAZOLE DYES

A small group of dyes of rather complex formula contain the
thiazole ring:

\ /s
c I
II c-

c II
/ \N

in which the indamine group is the chromophore. References to
these dyes in biological literature are rare, but the following have
been mentioned; two of them, it will be noticed are also azo dyes
and thus possess two chromophores.

f5

GERANINE G

C. I. NO. 117

S OH
CH3 /\/

C /-\ N = N /\/\
\/\ \ /

NaSOj

V

/

C24Hi6N304S2Na; MoL Wt. 497.509

[An acid dye)

This is a useful dye in fluorescence microscopy; see Pick (1935)
and Jenkins (1937).

FlO PRIMULIN C. I. NO. 81I

Synonym: Primuline yellow,
A mixture, consisting mainly of the following:

N N SOiNa

/\/ll /\/II I

CH, I I C\| I C\/-\ NH.

~\/\ I \/\ I \_/~

s s

CziHuNaOaSaNa; Mot. Wt. 475.523

{An acid dye)

Among various other thiazole dyes. Pick (1935) employed
primulin as a vital stain, for which it is useful because of its fluores-
cence under ultra-violet illumination. It has since become quite
widely used for the purpose.

Miscellaneous Dyes 231

Fl5 TITAN YELLOW G C. I. NO. 813

Synonym: Thiazol yellow.

S SOiNa SOiNa S

I
C

C28Hi9N506S4Na2; Mol. Wt. 695.706
[An acid dye)

This dye has been recommended as an indicator for determining
magnesium in body fluids. After removing the organic matter
and calcium from such solutions the dye is added; under alkaline
conditions it becomes red in the presence of magnesium and the
color observed can be compared with that of a standard previously
prepared.

Also, like the two preceding dyes, it has been employed in fluores-
cence microscopy; it is one of those recommended by Pick (1935)
and by Jenkins (1937) as a vital stain under ultra-violet illumina-
tion.

Fl7 THIOFLAVINE T C. I. NO. 815

Synonym : Thioflavine T G

CH3_/\_S

\ /~\_N_CH3
_ N=C-\_/ \

1\ CH3

CI CH3

Ci7 Hi9 No SCI; Mol. Wt. 318.861
{A basic dye)

Like the other related dyes here described, this one has proper-
ties as a fluorochrome; thus Metcalf and Patton (1944) have em-
ployed it, with ultraviolet light, in insect histology.

f20 THIOFLAVINE S C. I. NO. 8l6

{An add dye; a methylated and sulfonated derivative of primulin)

This is among the dyes that has recently come into use in con-
nection with fluorescence microscopy. Pick (1935) employed a
thioflavine as a vital stain, depending on its fluorescence under
ultra-violet light; probably he referred to this although there is also
a thioflavine T (C. I. No. 815). More recent authors (Jenkins,
1937, Levaditti, et al., 1940) definitely refer to thioflavine S, and

232 Biological Stains

the dye has now come to have a distinct recognition as a fluoro-
chrome. Thus Richards (1943) employs it in the demonstration
of the potato scab organism by ultraviolet light.

3. QUINOLINE DYES

Of the quinoline dyes only the following is of interest to bi-
ologists:

c5 PINACYANOL C. I. NO. 8o8

Synonym: Sensiiol red.

/ U -I \

I / I I \ I

~\ / \ r

CHj CH,— N-=CH CH CH- =N— CH^ . CHj

\
I

C25H25N2I; Mo^. WL 480.386
{A basic dye; absorption maxima [526 ^ 563] 608 in alcohol)

This dye is used primarily for color sensitization in photography,
but has been employed by Proescher (1933) for staining frozen
sections, and by Hetherington (1936) as a supra-vital stain for
mitachondria in blood. It has been employed in combatting
streptoccoci. S. H. Bensley (1952) has prepared a neutral stain
from it (an erythrosinate) and uses it for staining mast cells.

Another quinoline dye mentioned in biological literature is:
Quinoline yellowy C. I. No. 801.

4. DIAZONIUM AND TETRAZONIUM SALTS

Histochemistry has brought into microscopic use certain com-
pounds which are not dyes. Some of the histochemical reagents,
like basic fuchsin, are dyes used in decolorized state, and are dis-
cussed elsewhere in this book; others, like the oxidizing agents
periodic acid and tetra-acetic acid, are simple chemicals hardly
calling for special consideration. There are, however, certain im-
portant histochemical reagents which are so closely related to
dyestufls that they can hardly be omitted from these pages. They
are especially the diazonium, tetrazonium and tetrazolium salts.

The diazonium and tetrazonium salts have the same basic struc-
ture, and are considered in this section. They are salts of a
theoretical base having the structure R-N = N— OH [or
R-N(OH) = N]. Not only the base thus indicated, but its simple

Miscellaneous Dyes 233

salts of mineral acids are very unstable; they can often be stabil-
ized, however, by converting into zinc chloride double salts, and
those which are commercially available are ordinarily in this form.
In the formulae given below the unstable salt is listed in each case,
for the sake of simplicity. The diazonuim salts contain only one
— N = N+ group, the tetrazonium salts two. These salts are color-
less, but as the azo group (— N = N-), is a chromophore, their
transformation to colored compounds is easily effected. Their use
as histochemical reagents depends on the production of such
colored derivatives.

Among the papers introducing diazonium salts into histochemis-
try is that of Manheimer and Seligman (1948) who proposed a
method of demonstrating alkaline phosphatase in normal and
neoplastic tissues. This was shortly followed by an adaption of
the method to acid phosphatase (Seligman, 1949). The reagent
used in this work was:

a-NAPHTHOL DIAZONIUM CHLORIDE

NCI

Their histochemical applications are far from standardized, nor
is it certain yet which of them will be eventually preferred for the
purposes for which they are now employed in a rather experimental
way. All that seems advisable to do here is to list, with the for-
mula of the unstabilized salt, those that are known to be at
present commercially available.

DIAZO SALT OF P-CHLORO-O-NITROANILINE

NO2

/
Cl_/— \_N =: N— CI

Trade name: Red 3GS salt (Nat. Aniline)

DIAZO SALT OF 5-NITRO-2-AMINO NITROBENZENE

NO2

/

CH3_/~\_N = NCI
\_/

Trade name: Bordeaux GPS salt (Nat. Aniline)

DIAZO SALT OF 5-NITRO-2-AMINO METHOXYBENZENE

OCH3

/
02N_/~\_N=NC1

234 Biological Stains

Trade names; Echtrotsalz B (Holborn); Fast red salt B (Gurr);
Naphthanil diazo red B (Gurr); Kernechtrotsalz B (Gurr);
Nuclear fast red salt B (Gurr); Fast red B salt (Nat. Aniline)
See also nuclear fast red salt B in Chap. V (p. 70).

DIAZO SALT OF 4-NITRO-2-AMINO ANISOLE

OCH3

/

/— \_N=NC1

\_/
O2N—

Trade name: Scarlet RS salt (Nat. Aniline)

DIAZO SALT OF L-AMINO ANTHRAQUINONE

O N=NC1

/\_C_/\

O

Trade names: Naphthanil diazo red AL (DuPont); Fast red salt V
(Ciba); Red ALS salt (Nat Aniline).

The histochemical use of this reagent for demonstrating es-
terase, as well as a method for preparing it, as described by Nach-
las and Seligman (1949).

TETRAZO SALT OF O-ANISIDINE

OCH3 OCH3

\ /

C1N=N_/— \_/— \_N=NC1

Trade names: Naphthanil blue salt B, Fast blue salt B, Fast blue
salt BN (Gurr); Diazo blue B (DuPont); Blue BNS salt (Nat.
Aniline).

TETRAZO SALT OF 4,4-DIAMINODIPHENYLAMINE

C1N=N_/— \_NH_/-~\_N=NC1

Trade name: Black BS salt.

DIAZO SALT OF 6-BENZOYLAMINO-4-METHOXY-3-AMINO TOLUENE

O CH3

C — NH„/~\_N=:NC1
/

Trade name: Violet BN salt (Nat. Aniline).

Miscellaneous Dyes 235

DIAZO SALT OF AMINOAZOTOLUENE

CH3 CH3

/ /

N=N_/— \_N=NCI
\_/

Trade names: Fast garnet salt B (Gurr); Garnet GBC salt (Nat-
Aniline)

DIAZO SALT OF P-NITROPHENYL-AZO-2,5-DIMETHOXYAMINE

OCH3

/

02N_/— \_X=N_/~\_N=NC1

/
OCH3

Trade names : Fast black salt K (Gurr) ; Black K salt (Nat. Aniline)

5. TETRAZOLIUM SALTS

Among the important histochemical reagents, other than dyes,
are certain derivatives of tetrazole:

N\

II N
HC II

11 N

N/

A useful reference to consult concerning the chemistry of these
compounds and their biological applications is Smith, F. E.
(1951). The earliest prepared and best known of the derivatives
is 2,3,5-triphenyl tetrazolium chloride, which is commonly called

TETRAZOLIUM SALT

{Abbreviation TTC)

N = C"

/-\_ I I

\_/ N N

\ il
CI N

This reduces to the red compound, formazan, a compound con-
taining the azo-group:

N_C— ^

/-\ II I
\_/-N N

\ .
HN

236 Biological Stains

The red color produced on reduction was observed, and utilized
biologically, before the chemistry involved was understood, be-
cause the coloring thereby of certain parts of the embryo in seeds
was found to be an indicator of viability of the seeds. It was later
learned that other forms of living tissue could bring out the red
color; and histochemists discovered that the presence of certain
enzymes systems (dehydrogenases) could be visualized by their
ability to reduce the tetrazolium salt to formazan. See, for in-
stance, Pratt, Dufrenoy and Pickering (1948).

The compound has, therefore, found considerable histochemical
use; but it has certain drawbacks (among them photosensitivity)
which have led to investigation of other derivatives of tetrazolium.
Among them are:

2- (p-iODOPHEN yl) -3- (p-nitrophenyl) -5-phenyl

TETRAZOLIUM CHLORIDE

=N— N_/— \_N0.>

/-\_c \_/

\_/ \

CI— N=N /— \ I

This salt is less photosensitive than the preceding, and has the
additional advantage (Atkinson, Melvin and Fox, 1950) of slower
diffusion into ui>stained tissue.

NEOTETRAZOLIUM CHLORIDE

Synonym: Ditetrazolium chloride

/-\_c \_/ \_/) c_/-\

CI— N=N_/— \_/— \_N.=N— CI

The reduced form of this salt is deep purple to black, instead of
red as in the case of ordinary tetrazolium salt, which makes it
easier to distinguish in tissue. Antopol, Glaubach and Goldman
(1948) report that it stains malignant tissues rapidly and intensely.

DIMETHOXY NEOTETRAZOLIUM CHLORIDE

Synonym: Blue tetrazolium

C

,N— N_/— \ N— N,

C

Cl_N=N_/~\_/~\_N=N_Cl

\_/ \_/
CH3O/ \OCH3

Miscellaneous Dyes 237

This like the other tetrazolium salts is colorless itself, but oxidizes
into a colored formazan, known in this case as blue forniazan, be-
cause of its color. This deep blue pigment is a strong indicator
of certain types of enzymatic activity, but is much more toxic
than ordinary tetrazolium chloride. See Smith (1951).

6. MINERAL PIGMENTS

' There are a considerable number of pigments of non-organic
nature, which are much better known to the manufacturer of
paints than to the biologist. They are in no sense dyes; but a few
of them are occasionally used in microscopic work or for some other
biological purpose. They were not listed in the first editions of this
book because they were regarded as lying outside the field covered.
The following three, however, are occasionally mentioned by the
biologist; and they seem worth mentioning, if for no other reason,
to keep them from being confused with the dyes. Of these three,
the first one is generally known for what it is; but the other two
are more apt to be ordered as though they were dyes instead
of mineral salts.

PRUSSIAN BLUE C. I. NO. 12.88

Synonyms: Berlin blue. Chinese blue. Paris blue. Milori blue.

Steel blue.

The well-known colored salt, ferric ferrocyanide, Fe4(FeC6N6)3,
finds its greatest use in the manufacture of paints, but it has oc-
casional biologic employment, chiefly for coloring anatomical
specimens, for demonstration. of the circulatory system by means of
injection. It is mentioned in connection with the Gomori (1936)
technic in which K4Fe(CN)6 is used as a microchemical reagent for
iron in tissues; by this procedure Prussian blue is formed in situ in
the tissues.

SKY BLUE C. I. NO. 12.86

Synonyms: Coelestin blue. Coeline. Coeruleum.

This pigment is essentially cobaltous stannate, CoOSn02. It is
called for primarily as a constituent of artists' oil or water colors,
but has been given occasional mention in biological literature.

RUTHENIUM RED

An ammoniated ruthenium oxychloride prepared by making an
ammoniacal solution of ruthenic chloride, RuCla. It has been
employed, with or without the addition of acetic acid, in micro-
scopic work, primarily as a test for pectin. It is thought by some
to be specific for this purpose.

238 Biological Stains

It should also be mentioned that the name emerald green, listed
on p. 142 as a synonym of brilliant green, should more correctly be
applied to a hydrated chromium oxide; while Victoria green is a
mixture of this compound with two other mineral pigments —
although mentioned elsewhere in this book (p. 141) as a synonym
of malachite green. These two pigments seem to have no biologic
use, but are mentioned here to avoid possible confusion with the
two dyes to which the same names have been given.

CHAPTER XI
NEUTRAL STAINS

THERE are two ways in which dyes may be compounded. In
the first place it is possible to mix mechanically any two dyes,
and if they are of different colors with different selective powers,
double staining effects may be procured. In the second place, it is
often possible to form a chemical union between two dyes and thus
to obtain an entirely new compound which may have quite strik-
ing staining properties. It is such compounds as these, rather than
simple mechanical mixtures, that are ordinarily referred to as
compound dyes.

The simple anilin dyes, it will be recalled (see Chapter II), owe
their properties as dyes or as biological stains to the basic or acidic
character of the dye molecule. Those parts of the protoplasm which
are acid in nature (e. g., chromatin) tend to react with the basic
dyes and to be colored by them; while those which are basic
(e. g., cytoplasm) react similarly with the acid dyes. (This, to be
sure, is not the whole theory of staining, as the process is quite
complex and involves physical and mechanical factors as well; but
it serves to illustrate the difference between the two kinds of stains.)
Now, as already explained, the dyes are not used as free acids or
free bases; but rather as sodium or potassium salts of the acid
dyes, and as chlorides (or salts of some other colorless acid) of the
basic dyes.

It is well known that when two salts, such as sodium chloride
and ammonium nitrate, are mixed in solution there is an inter-
change of ions (metathesis), and the resulting solution, when it
reaches equilibrium, contains not only the original salts but also
the four free ions and the two alternate compounds as well, in this
case sodium nitrate and ammonium chloride. Now if one of these
two new compounds happens to be insoluble, as silver chloride for
example, which would have been formed if silver nitrate had been
substituted for ammonium nitrate, it is thrown out of solution,
and equilibrium is not reached until the solution is free (or at least
practically free) from the two ions which are insoluble in combina-
tion. In the same way, when a sodium salt of an acid dye and a
chloride of a basic dye are mixed in solution, there is a similar
tendency for the ions to interchange. Ordinarily the dyes are
weaker acids and bases than the chlorine and sodium ions respec-
tively; and if the compound dye formed were soluble in water
there would be little chance for much of it to be produced. As a
matter of fact, however, it is generally insoluble and is therefore
thrown out of solution; hence the compound dye can be formed
in considerable quantity.

239

240 Biological Stains

Considering that salts of color acids are called acid dyes, and
salts of color bases are known as basic dyes, a theoretically satis-
factory name for the compound dyes in which a color acid is com-
bined with a color base would be "neutral dyes". The term is not
ordinarily used this way, however; in fact, the dye chemist uses
the same term in an entirely different sense. The chemist has not,
however, pre-empted the term "neutral stain"; and considering
that this class of compound dyes is employed for biological stain-
ing rather than general dyeing purposes, the name is quite ap-
propriate. The Stain Commission, therefore, has adopted the
use of the term neutral stain for this purpose and defines it as a
compound of an acid dye and a basic dye in which both the anion
and the kation contain chromophore groups.

Ehrlich (1898) introduced a different term, "tri-acid dyes",
which he intended, apparently, to apply to the whole class of
neutral stains. He seems to have introduced this term on the
basis of two assumptions; first, that most of the basic dyes are
triamino compounds (and therefore potentially tribasic); second,
that in the ordinary mineral acid salts of these dyes only one of the
affinities for acid is satisfied, but when compounded with an acid
dye the other two affinities also become satisfied. The latter
assumption, at least, seems to be entirely incorrect. As just ex-
plained, the reaction between an acid dye and a basic dye seems to
be purely one of metathesis, and there is no reason whatsoever
for assuming that the dye anion attaches itself to any other place
in the basic dye molecule than that previously occupied by the dis-
placed mineral anion. Ehrlich's term, however, has persisted for
the particular "tri-acid stain" which he specially recommended
(see below); and since this particular neutral stain is no more a
tri-acid product than any other, some explanation of the term
seems necessary.

It is possible to obtain an endless variety of such dyes; but in
practice only a certain number of them have proved useful. Among
the basic dyes the most suitable for this purpose are the thiazins
and the rosanilins (which act as strong ammonium bases) ; among
the acid dyes, the eosins and the sulfonic acids (e.g., orange G and
acid fuchsin).

Although the neutral stains are insoluble in water, they are sol-
uble to a greater or less extent in excess of either the acid or the
basic dye. Thus if an aqueous solution of acid fuchsin is neu-
tralized by adding drop by drop an aqueous solution of methyl
green, there is at first no precipitation, because the methyl green
salt of acid fuchsin is kept in solution by the excess of acid fuchsin.
After the proper amount of methyl green has been added, however,
and the mixture has stood long enough for the reaction to take
place, the neutral stain is precipitated and the solution becomes
nearly colorless. Then if more methyl green is added the neutral

Neutral Stains 241

stain is slowly dissolved again; but as a rule neutral stains are less
soluble in excess of basic dye than in excess of acid dye.

As simple aqueous solutions of these compound dyes are im-
possible and as alcoholic solutions of dyes do not stain well,
various methods are employed to secure their action on the tissues.
In some instances they are kept dissolved by the presence of an
excess of acid or base (particularly the former) ; in others a certain
quantity of acetone or methylal is used to hold the neutral stain in
solution; sometimes (as in the original Romano vsky stain) the
compound dye is used immediately after mixing, before the re-
action is complete or precipitation has taken place; or again (as in
the Wright stain) methyl or ethyl alcohol may be used as a solvent,
and then after applying the alcoholic solution to the slide it may
be diluted with water. This latter method is particularly effica-
cious, possibly because a temporary state of supersaturation
occurs on addition of the water, and there are other known in-
stances where such a state enhances staining power. It should
be remarked that when water is mixed with such an alcoholic
solution, dissociation must take place and an unstable equilibrium
must exist until the compounds insoluble in the mixture of water
and methanol have been precipitated; under such circumstances
the staining reactions may be rather complicated.

It is assumed that these compound dyes act on the protoplasm
somewhat as follows: certain parts of the cell have an affinity for
the neutral stain and take it up as such; others, having an affinity
for the basic dye, break up the neutral stain so as to obtain the
basic portion of it, or if dissociation has taken place, take up the
basic ion directly; while other parts of the cell with an affinity for
acid dyes similarly combine with the acid portion of the stain.
These three types of cell structures are known as neutrophile,
basophile and oxyphile elements, respectively. The differentia-
tion thus produced gives the neutral dyes their great value.

Ehrlich's "Tri-acid Stain."

The first neutral stain proposed for microscopic work was the
"tri-acid stain" of Ehrlich (1910, II, 313). In forming this com-
pound dye, acid fuchsin and orange G are mixed in solution and to
the mixture is then added such a quantity of methyl green that
there is still an excess of the acid dye. This excess of the acid dye
allows the neutral stain to stay in solution. The dye thus formed
is a useful blood stain, and brings out finely the different structures
in the leucocytes.

The explanation of the name of this stain (which was based on a
mistaken chemical theory) is explained on a preceding page.

Slight modifications of the stain have been used for tissues. The
best known is that of Biondi and Heidenhain. (See Krause, 1926-7,
p. 457, or Lee, 1937, p. 166).

242 Biological Stains

Thiazin Eosinates*

The first worker to combine eosin and methylene blue was
Romano vsky (1891). He realized that a mixture of these two
dyes had great selective properties as a stain, and showed it to be
excellent for blood, particularly in bringing out the malarial para-
site. He also appreciated that it was more than a mere mixture of
the two dyes and that some new dye having the property of giving
the nuclei a red color was present. It was some time later before
the nature of this new dye was known, although it was subsequent-
ly named azure I or methylene azure; its true chemistry has
scarcely been understood until recently (see p. 107). Methylene
violet, and "methylene azure", which probably was also present,
had already been described by Bernthsen (1885). How these
new dyes were formed in the Romanovsky stain was not known
then; although Romanovsky stated that different lots of methylene
blue solution varied in their ability to give a good blood stain, and
that old solutions on which a scum had formed were best.

One interesting point in connection with this early work is that
Bernthsen made the statement that the absorption maximum of
Azure I is at 650 m/z. This statement indicates, as can readily
be verified by consulting the absorption data of the azures given
in Chapter VI, that his azure I may have been primarily azure B.
In much recent work concerning the chemistry of azure I, this
early statement of Bernthsen's seems to have been overlooked.

Present day blood stains are often spoken of as modified Roman-
ovsky stains; although the modifications are so great as to make
them of a very different nature. The first modification was made
by Nocht (1898) who concluded that the differential staining was
due to the formation of other dyes by the decomposition of meth-
ylene blue. Unna (1891) had already described what he called
polychrome methylene blue, made by heating a solution of meth-
ylene blue on a water bath with potassium carbonate. Nocht
decided to use this in the Romanovsky stain instead of untreated
methylene blue. He found that it gave very good results if prop-
erly neutralized before mixing with eosin; and then learned that
better results could be obtained by the use of a smaller amount of
alkali and a longer period of polychroming, without subsequent
neutralization.

The next step in preparing blood stains was made by Jenner
(1899) who collected the precipitate formed when methylene blue
and eosin are mixed, and redissolved it in methyl alcohol. He did
not use polychrome methylene blue, and his stain lacked the
nuclear staining principle of Romanovsky 's and Nocht's stains
unless allowed to stand for some time before using; but it was an
important step in that he showed the possibility of collecting the

*An account of the history of these blood stains is given by MacNeal (1906),
and by Conn et al. (1933).

Neutral Stains 243

precipitated compound stain and of dissolving it in some solvent
other than water. Jenner's stain is still called for occasionally.
Although it is markedly inferior to the modern type of blood
stains when used fresh, it does polychrome on standing in methan-
olic solution, and this fact undoubtedly explains its continued use.

Combining Jenner's procedure with the Nocht stain was the
next logical step and was taken independently by Renter (1901)
and by Leishman (1901), The method thus introduced was
briefly to foUow Nocht's technic of combining eosin with poly-
chrome methylene blue, but then to filter off the precipitate and
to redissolve it in methyl alcohol, not adding further water until
the moment of applying the stain to the blood films.

Modern blood stains are in general modifications of Leishman 's,
differing only in detail. Wright's modification (1902), the one
most used in America, (see Mallory and Wright, 1924, p. 170)
differs from Leishman's only in that he prepared polychrome
methylene blue by heating for only an hour in flowing steam,
whereas the Leishman technic calls for twelve hours at 65°C, with
subsequent standing for ten days.

Previous editions of this book have contained directions for pre-
paring Wright stain. It is, in fact, perfectly possible for a labora-
tory worker to prepare a blood stain of this type that gives excel-
lent results; and the technic of doing so is not specially difficult
to one who has the necessary experience. Nevertheless, it has
proved that the directions given have not yielded uniformly de-
pendable results. It is desirable to have exactly the right amounts
of acid and basic dye present to combine without much excess of
either; and the relative amounts called for on theoretical grounds
do not always yield best results in practice. Moreover, spectro-
photometric control during polychroming seems to be necessary
to attain a uniform product, and the average laboratory worker
does not have the necessary facilities. Furthermore, this stain,
either in powdered form or dissolved in methyl alcohol, may be
purchased from stain companies at present, usually more uniform
in quality than can be made up by the user. Accordingly,
directions for its manufacture seem hardly called for here.

Balch's modification calls for a polychrome methylene blue pre-
pared by standing 10 days with precipitated Ag20. Hastings
modification (1905) differs from that of Wright or Leishman in
that the polychrome methylene blue is neutralized with acetic
acid, and an unpolychromed solution of methylene blue is mixed
with it before eosinating. It is interesting, as a matter of fact, to
note that the method of preparing Wright stain followed by some
stain manufacturers is more like that of Hastings than like that of
Wright. In general American manufacturers sell the same prod-
uct as "Wright stain" and as "Hastings stain"; perhaps it should
all be known by the latter name. Lillie (1944a) has recently
suggested the use of a methylene blue polychromed by heating

244 Biological Stains

with a definite proportion of K2Cr207 in acid solution, a process
which he finds to yield a more uniform product.

Giemsa's and MacNeal's modifications are somewhat different.
In order to start with a more definite compound than polychrome
methylene blue, Giemsa (1902) used more carefully controlled
methods of oxidation and obtained a product which he considered
to be the same as Bernthsen's Azure I. This he combined with
eosin Y, to obtain a more definite compound than in the case of
stains of the Leishman type. Subsequently he added methylene
blue to the azure before combining with the eosin, and in that way
secured better differentiation. He did not state his method of
preparing azure I, but apparently gave it secretly to Dr. Grubler's
Laboratory, which subsequently became K. Hollborn and Sons;
and the latter company made much of this "trade secret" in
claiming their Giemsa stain to be the only authentic product of
that name. Besides azure I, this company put on the market an
azure II which was a mixture of Azure I and methylene blue in
equal parts. The eosinate of this mixture, known as azure-II-
eosin, was the chief ingredient of Giemsa stain. There probably is
no real secret involved in the composition of these azures. Giemsa
stated that he used Bernthsen's methylene azure, and Bernthsen
described this as having an absorption maximum at 650 m^t; as this
is approximately that of present day azure B, we can assume that
Giemsa either had the latter or a mixture of higher and lower
homologs having optical properties similar to azure B. There has,
however, been considerable confusion on this subject of recent
years; and in part this has grown out of certain erroneous in-
ferences derived from MacNeal's excellent work (discussed below)
on these stains.

The formulae recommended by Giemsa for preparing solutions of

this stain are:

For blood For tissue

Azure-II-eosin 3- g- 3. g.

Azure II 0.8 g. 0.8 g.

Glycerol, c.p 250 g. or 200 ml. 125 g. or 100 ml.

Methyl alcohol, neutral, acetone free 250 g. or 312 ml. 375 g. or 457 ml.

There is apparently a reason for the quantity of glycerin called
for in the first of these formulae (50% by weight or about 40%
by volume). Recent work in one of the Stain Commission labora-
tories indicates at least 40% by volume to be necessary to give a
reasonably stable solution.

Substituting American dyes for those in the old Giemsa formula,

and following the recommendations of Lillie, the following seems

advisable :

Azure A eosinate 0.5 g.

Azure B eosinate 2.5 g.

Methylene blue eosinate . . . 2.0 g.
Methylene blue chloride ... 1.0 g.

Glycerol 375. ml.

Methyl alcohol, reagent. . . .375. ml.

Neutral Stains 245

Mixed powdered dyes of approximately this formula are available
from some of the American manufacturers.

MacNeal (1922) proposed a stain, intended to give similar re-
sults, although prepared on different principles. He showed that
by mixing Bernthsen's methylene violet and azure I with meth-
ylene blue and eosin Y in definite proportions, dissolving in
methyl alcohol, and employing like a Leishman or Wright stain,
one could obtain results almost identical with those secured by
these other blood stains without the uncertainties introduced by
their methods of manufacture. Soon after publishing this paper
he realized that methylene azure could be a mixture and certainly
was by the methods he had used in preparing it; (MacXeal, 1925).
He showed it to contain both azure A and azure B, whose chem-
istry is discussed in Chapter \ I of this book. He regarded the
former as the more important ingredient, and stated that the stain-
ing effects of Azure B are little different from those of a mixture
of azure A and methylene blue. This statement of his gave rise
to the impression that azure B was not a satisfactory stain; and
only recently has this impression come to be corrected in the minds
of users of these stains. MacNeal's tetrachrome stain is ordinarily
made up according to the following formula:

Methylene blue chloride (dye content about 90%) 1-0 g.

Azure A 0.6 g.

Methylene violet, Bernthsen, free base 0.2 g.

Eosin Y, (dye content 80-85%) 10 g.

It will readily be seen that the dry tetrachrome stain is a mixture
rather than a chemical compound like the Leishman stain and its
modifications. The four dyes mixed together combine but very
slowly even after dissolving in the alcohol; and the first pro-
nounced chemical reaction occurs only after diluting with water in
actual application. There is evidence, however, that certain chemi-
cal reactions (and of an undesirable nature) take place in the
alcoholic solution, and thus interfere with the keeping qualities of
the solution. Manufacturers of tetrachrome stain realize this and
give directions for allowing the alcoholic solution to stand 48
hours at 50°C and then filtering; this practice seems to remove
whatever harmful compound may have formed, and the solution
thus obtained is reasonably permanent. Undoubtedly, more-
over, methyl alcohol alone is an undesirable solvent for this mixture
of dyes, just as it is in the case of Giemsa stain. There can be little
question but that the addition of 40% glycerol, by volume, as
recommended by Giemsa, would make the solvent for the tetra-
chrome stain more satisfactory and would improve the keeping
qualities of the solution.

It can readily be inferred that the thiazin eosinates, made up by
any of the above mentioned procedures, must be very complex
mixtures. Theoretically each of the azures or other oxidation

246 Biological Stains

products of methylene blue present in the polychromed solution
may combine individually with the eosin, thus yielding several
different eosinates in the precipitate. Unless the precipitate is
washed with two or three changes of water it usually contains a
considerable excess of one or the other of the uncombined dyes, and
this may further increase the complexity of the mixture. If
properly washed, the precipitate should be free, or almost free, of
uncombined basic or acid dye, because of their great solubility,
and possibly such is also the case in the methanolic solution of the
precipitate; but it is certain that when water is added in the process
of staining, dissociation must occur with the formation of the
acid and basic dyes from which the compound was derived. This
is another contribution to the complexity of the solution with
which the staining is accomplished. Complex as the mixture is,
Lillie (1942) has shown that it can be quite effectively analyzed
spectrophotometrica,lly ; and that from the position and width of
the absorption bands, one can get some idea as to the composition
of the polychrome methylene blue from which the eosinate was pre-
pared. By comparing such spectrophotometric results with the
behavior in staining, one can obtain practical information as to the
value of the different azures in a stain of this type.

In addition to the chemistry of the constituent dyes, another
important factor in a blood stain of this type is the nature of the
solvent. Methyl alcohol, as explained above, is the usual solvent
employed; but there are many grades of this reagent available,
and not all are equally suitable for the purpose — a fact which at
one time was not fully appreciated. Apparently absolute purity of
the methanol is not needed; but one point is very important — the
solvent must be neutral in reaction. Methanol, as formerly pre-
pared by the destructive distillation of wood, often contained con-
siderable amounts of the other two major products of that decom-
position, acetone and acetic acid. The presence of acetone seems
to be unimportant (in spite of previous recommendations that
methanol for blood stains should be acetone-free); acetic acid,
however, is quite deleterious in that it tends to precipitate eosin
as its color acid, when the methanolic solution is diluted with
water. Special methyl alcohol for blood stains is on the market,
but the modern synthetic methanol of reagent grade proves quite
satisfactory; its further purification with Ag20, as sometimes
recommended to free it from aldehydes and ketones, seems to be
quite unnecessary. (See Lillie, 1944b).

The two blood stains for which there is now most demand in
America are Wright stain and Giemsa stain, while the tetra-
chrome stain is used less frequently. Wright stain and the
tetrachrome stain both give almost identical results and are
handled very similarly. If one is to prepare the stain himself,
there are reasons for preferring the tetrachrome stain as it can be

Neutral Stains 247

made up with more certainty of obtaining a satisfactory product.
Ordinarily today, however, one purchases his blood stains already
prepared by some manufacturer; and the Wright stain on the
market seems to be as reliable as the tetrachrome stain.

Giemsa stain, although originally intended to be more definite
in composition than other blood stains, is possibly the most com-
plicated of them all. As a simple stain for blood in thin smears,
some find it to have no distinct advantage over stains of the
Leishman-Wright type, although others claim it to show greater
purity of color and sharpness of definition, with deeper staining of
chromatin; such advantages as it has for this purpose are offset
by the longer staining time and larger volume of solutions required.
As a stain for malarial blood, however, designed to show the
malaria plasmodium, Giemsa stain is definitely preferred. In
searching for the malaria organism the present approved technic
calls for the use of thick smears of blood in which laking of the red
corpuscles is brought about by distilled water (either as a diluent
of the stain or placed on the smears before staining) ; accordingly a
stain for the red cells* is not required, but one is necessary that
brings out the leucocytes and the plasmodia. For this, a prop-
erly prepared Giemsa stain has decided advantages over Wright
stain. Good results have quite consistently been obtained with
the German Giemsa stain, when it has not become unavailable
because of war; and there is good reason to believe that it has been
quite constant in composition. As remarked above, its manufac-
turers used to lay considerable stress on the /'secret" character
of azure I and to claim that since they alone know that secret, no
other Giemsa stain except theirs could be reliable. As mentioned
above, however, no real secret seems to have been involved; and
recent investigations have shown that a Giemsa stain, having one
of its absorption maxima at about 655, as recommended by Lillie
(whether prepared in the laboratory or put on the market as a
commercial product) is an excellent malarial stain, as judged by
technicians familiar with the technic in question.

During the two decades from 1920 to 1940, however, much
Giemsa stain was on the market in America which did not conform
to the requirements now specified by Lillie. This was largely due
to the stress laid by MacNeal on Azure A as the important in-
gredient of azure I, and to the fact that Holmes and French later
(1926) said quite definitely that azure B has no staining value. It
took some time to counteract the effect of such statements, and
meanwhile American manufacturers of Giemsa stain, with the
endorsement of the Stain Commission, used azure A, without any
appreciable azure B, in its preparation. Since Lillie has called
attention to the importance of azure B, however, the practice of
the stain companies has changed; and now there is an American

248 Biological Stains

Giemsa stain available which gives results very much like the im-
ported product.

Other uses of thiosin eosinates. Although thiazin eosinates are
specially known for their use as blood stains, they are coming more
and more to be employed for other purposes. Tolstoouhov (1928),
for instance, recommended a Romanovsky mixture as a tissue
stain, his solution proving stable because of the excess of methylene
blue present; he showed that such a mixture can produce a variety
of staining effects according to the reaction given to the solu-
tion by adding varying quantities of N/100 NaOH or HCl. The
same author (1929) later proposed a similar mixture for differ-
entiating one kind of bacteria from another. Churchman (1933)
has employed Wright stain for demonstrating bacterial capsules,
and Dutton (1928) to stain bacterial spores. Giemsa stain has
been used similarly, and also as a tissue stain. Lillie and Paster-
nack (1932, 1936) have, in fact, shown that various thiazin
eosinates can be used very effectively as a tissue stain; Lillie (1941)
has given in detail a technic for staining tissue with a specially
modified Wright stain following hematoxylin. His latest recom-
mendations (1944c) call for azure A, azure C or toluidine blue
salts of eosin Y or eosin B, with special preference for those of
eosin B which are stated to give deeper red staining of hemoglobin
and granules.

PROCEDURES RECOMMENDED BY THE COMMISSION IN WHICH
THIAZIN EOSINATES ARE USED*

PAGE REFERENCE TO

NAME OF PROCEDURE

Staining Procedures

Buffered azure eosinates for tissue

LV17

Wright stain for blood smears

ID3-3

MacNeal's tetrachrome stain for blood smears

ID3-5

Giemsa stain for thin films

ID3-6

Giemsa stain for thick films

ID3-6

Giemsa or Wright stain for bone marrow smears

ID3-IO, 11

*Under this heading are given references to procedures described in detail in
Staining Procedures, edited by Conn and Darrow (1943-4).

Other Compound Stains

Various other compounds of acid and basic dyes have been used
for special purposes. The basic dyes employed in these com-
pounds most commonly are perhaps methyl green and methylene
blue; but sometimes basic fuchsin, pyronin or rhodamine or even
a weak base like neutral red is used. Most common among the
acid dyes in these compounds are eosin, orange G and acid fuchsin ;
but certain others are occasionally employed. Picric acid forms a
few useful compound dyes, rosanilin picrate (i. e. the compound

Neutral Stains 249

of basic fuchsin and picric acid) being especially well known as a
tissue stain.

The Pappenheim panoptic triacid stain is a modification of
Ehrlich's triacid compound. In this combination methylene blue
or methylene azure is substituted for methyl green. It is a tissue
stain of use in certain special technics. Another well-known
neutral stain is the Twort (1924a and b) formula in which neutral
red and light green are combined. The Twort stain is valuable
for staining animal parasites and other animal organisms in tissues.
Maurer and Lewis (1922) (see page 165) have combined safranin
in a neutral stain with some acid violet and have employed the
compound dye in staining glandular tissue.

Ehrlich proposed various other neutral stains, the best known
being a compound of acid fuchsin and methylene blue used
for staining blood; and a compound of narcein, an acid dye, with
two basic dyes pyronin and methyl green or methylene blue. A
combination of orange G with a gentian violet has been proposed
by Bensley (1911) as a stain for the A and B cells in the islands of
Langerhans.

APPENDIX I
TABLES RELATING TO STAINS

How TO Use These Tables

Identifying a dye from its index number. Dyes in England and the United States
are generally designated by their Colour Index number, in Germany and several
other countries by their Schultz number. If the C. I. number alone is known for
one of the biological stains, it can be identified by Table 1, in which the dyes are
arranged in the order of the C. I. nmnbers; if known by the Schultz number it can
be identified by Table 2, in which the Schultz numbers of these same stains are
arranged serially, followed by the corresponding C. I. numbers and page references
to where the dyes are described in this book.

Table 1 can also be used in studying the synonymy of the stains; but will not
prove as convenient as the general index for identifying a dye from some synonym,
as the arrangement is not alphabetical.

Learning the common uses of stains. In Table 3 the dyes are listed under their
preferred designations in the order in which they occur in the main part of this
book. In the second column are given their principle uses as stains. References
to the authors of the procedures are not given in this table, but can be obtained by
consulting the page in the main part of the book where the dye in question is
described.

Selecting stains for some general field of work. Table 5 includes lists of the dyes
most commonly used in general fields of microscopic work, e.g. animal histology,
plant histology, etc. It should prove useful in selecting the stains needed in
equipping a new laboratory in some one of these fields.

Learning the solubility of a stain. In making solutions of stains it is often de-
sirable to know their solubility in either water or 95% alcohol. Figures indicating
these solubilities of recrystallized dyes are given in Table 6, which includes all the
most commonly used stains, and numerous others; the arrangement in this table
is alphabetical. The solubilities of commercial dyes, however, vary widely from
these figures for the purified products; the former are ordinarily less soluble, though
occasionally the opposite. A limited amount of information is available, thanks
to the cooperation of the American Pharmaceutical Association, as to the solubil-
ities of commercial samples of about 25 of the commonly used stains; this is given
in Table 7, and in some cases may prove more useful in making up a staining fluid
than that given in Table 6.

251

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Tables Relating to Stains

257

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Tables Relating to Stains

259

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Tables Relating to Stains

261

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202 Biological Stains

Table 3. The Principal Uses of the Most Important Stains

Name of stain
and page
reference*

Application

Naphthol

green Y

p. 65

With gentian violet and azocarmine G; as tissue stain.

Naphthol

green B

p. 66

With orcein, iron-hematoxylin and azocarmine G; as
tissue stain.

For cytoplasm. Contrast stain. Often in solution with
anilin blue W.S.

Picric acid

With acid fuchsin; Van Gieson's connective tissue stain.

With crystal violet; in cytology of fungi.

Staining cell walls of marine fungi.

Martins yellow
p. 68

Pianese's stain for cancer tissue; with acid fuchsin and
malachite green.

Same technic adapted for staining pathologic plant tis-
sue (fungus diseases).

In contrast to resorcin blue for staining pollen tubes in
styles.

For light filters in photomicrography.

Aurantia
p. 68

Champy-Kull technic for mitochondria; with acid
fuchsin and toluidine blue.

In studying polyploidy in plants.

For bone sections.

Fast yellow
p. 70

Combined with eosin, phloxine and anilin blue; in the
study of chromolysis by Unna.

With hematoxylin and acid fuchsin; as tissue stain.

Oil yellow II
p. 71

As carcinogenic substance.

A general cytoplasm stain; a good substitute for Bis-
marck brown.

Chrysoidin Y
p. 71

Vital staining of pollen tubes.

Vital staining of insects.

Staining Golgi apparatus.

*The pages listed in this column refer to the present book.

Tables Relating to Stains
Table 3. (Continued)

263

Name of stain
and page
reference*

Application

Benzene-azo-

a-naphthylamine

p. 72

Vital stain for protozoa

A valuable plasma stain in sections of tissue.

Background stain for hematoxylin and other nuclear
stains in botanical histology.

As cytoplasm stain, in contrast to crystal violet and
safranin; Flemming triple stain.

Orange G
p. 72

For cytoplasm and red blood cells in Mallory's connec-
tive tissue stain; with anilin blue and acid fuchsin.

For tissues; with methyl green and acid fuchsin in
Ehrlich-Biondi-Heidenhain technic.

Ehrlich's "tri-acid" stain for blood; combined with

Chromotrope 2R
p. 73

Azophloxine GA
p. 73

Sorbine red
p. 74

Azo acid blue B
p. 76

methyl green and acid fuchsin.

Combined with gentian violet as Bensley's "neutral
gentian"; for islands of Langerhans.

Counterstain to thionin; in plant pathology.

As constituent of Kornhauser's "Quad" stain.

Counterstain to celestin blue; in animal histology.

As counterstain in neuropathology.

Substitute for "xylidine ponceau" in Masson trichrome
technic.

Staining red blood cells in sections.

Substitute for "xylidine ponceau" in Masson trichrome
technic.

Staining nervous tissue.

Nitrazine
p. 76

Sudan II
p. 76

Ponceau 2R
p. 77

As substitute for xylidine ponceau in Masson trichrome
stain.

Fat stain for central nervous tissue, in supersaturated
isopropanol solution.

In Masson's trichrome tissue stain.

264

Biological Stains
Table 3. (Continued)

Name of stain

and page

reference*

Application

Oil brown D
p. 77

As fat stain in Lillie's supersaturated isopropanol solu-
tion.

Kr^rritfifni Y i"Ari

For cytoplasm, before staining with Heidenhain's hema-
toxylin.

p. 78

For sections of spleen, testis and liver; with methyl
green and thionin.

Substitute for "xylidine ponceau" in Masson trichrome
technic.

Sudan R
p. 78

Probably the dye employed in Weichherz's modification
of the Kahn test for syphilis.

JTsiniiQ (TVf^f^'n R

For vital staining of chondriosomes; also of fungi and
protozoa.

p. 78

For sections of embryos; with neutral red.

Sabins method for supravital stain for blood.

Janus black
p. 80

Vital staining of pollen tubes.

Metanil yellow
p. 80

In Masson's trichrome tissue stain.

Methyl orange
n 81

For determining reaction of cell sap in plants.

Witjb crystal violet in staining pollen tubes.

Prontosil
p. 81

Vital stain for insects and plants.

Brilliant yellow S
p. 82

As counterstain to carbol fuchsin in staining acid -fast
bacteria; Doglio's method as interpreted by Mar-
golena.

Orange I
p. 82

With malachite green for sections of plant tissue.

As cytoplasm stain; with eosin and azure C.

Orange II
p. 83

In place of orange G in the Ehrlich-Biondi stain.

For keratin in sections of skin.

In Kalter's quadruple stain, with fast green FCF, safia-
nin and crystal violet.

Nareein
p. 83

As component of some of Ehrlich's neutral stain mix-
tures.

Tables Relating to Stains
Table 3. (Continued)

265

Name of stain

and page

reference*

Application

Amaranth
p. 84

For axis cylinders.

Staining cells in tissue culture.

In color photography.

Methyl red
p. 84

As vital stain for protozoa.

Thiazine red R
p. 85

As counterstain in animal histology, especially with
hematoxylin, for skeletal and cardiac muscle.

Naphthol blue black
p. 85

By Lillie as a stain for collagen.

Brilliant croceine
p. 86

By Lillie, as substitute for "xylidine ponceau" in Mas-
son's triple stain.

Sudan III
p. 86

For fat tissues; Daddi-Herxheimer technic.

For differentiating suberized and cutinized tissue in
plants.

Sudan IV
p. 87

For fat in tissues, Michaelis-Herxheimer technic, and
Lillie's supersaturated isopropanol method.

Oil red

n 88

For staining fat; as substitute for Sudan III or IV.

With pyridin; for staining fat.

Oil red 4B
p. 88

As fat stain, in Lillie's supersaturated isopropanol solu-
tion.

A general plasma stain.

For cytoplasm, after polychrome methylene blue or
Unna's hematein.

Biebrich scarlet,
W. S.
p. 89

Mixed with alum hematoxylin; for staining tissue sec-
tions.

In neutral stain combination with ethyl violet, by Bowie,
for staining islets of Langerhans.

As substitute for xylidine ponceau in Masson trichrome
stain.

With orange G and fast green FCF, for staining vaginal
smears.

In "picro-Mallory" combination of McFarlane.

266

Biological Stains
Table 3. {Continued)

Name of stain
and page
reference*

Application

Ponceau S
p. 89

By Curtis (more recently Ruth; Leach) as an improve-
ment over acid fuchsin in the Van Gieson connective
tissue stain.

Orseillin BB
p. 90

In contrast to crystal violet in staining a mycorrhizal
fungus.

By Maneval, as bacterial stain.

Bismarck brown Y
p. 90

A general plasma stain, formerly much used. A good
mucin stain. Good for vital staining and for staining
in bulk.

For bacteria, particularly in contrast to gentian violet
in the Gram technic.

Chromosomes in smear preparations of eggs.

Background stain for hematoxylin and other nuclear
stains.

By Griesbach for axis cylinders.

By Schaffer for sections of embryos.

For staining plant mucin.

Congo red
p. 92

By Klebs as reagent for cellulose.

By Blackman as stain for Uredineae.

By Matsuura for staining elastic tissue.

By Benians as negative stain for bacteria.

By Merton for protozoa.

By Gutstein as vital stain for yeasts.

By Bennhold for amyloid.

Erie garnet B
p. 93

Mixed with azure A; by Geschikter as stain for frozen
fresh tissue.

Trypan red
p. 93

For vital staining.

An early chemotherapeutic agent.

Benzopurpurin 4B
p. 93

Plasma stain, especially in contrast to hematoxylin.

Tables Relating to Stains
Table 3. (Continued)

267

Name of stain
and page
reference*

Application

Brilliant purpurin R
p. 94

By Gutstein as vital stain for yeasts.

By Lillie in connective tissue stain, with azofuchsin G
and naphthol blue black.

Vital red
p. 94

By Evans for vital staining.

Vital staining of Protozoa.

Azo blue
p. 95

As substitute for india ink in demonstrating bacterial
capsules.

Histochemical demonstration of magnesium (with
quinalizarin and Titan yellow).

Dianil blue 2R
p. 95

For vital staining.

Trypan blue
p. 96

For vital staining.

Evans blue
p. 96

For vital staining.

Vital new red
p. 96

As vital stain.

Niagara blue 4B
p. 97

By Varrelman for demonstrating vascular system of
plants.

Occasional use in general histology, and possibly in vital
staining.

Chlorazol black E
p. 97

In general histology.

As auxiliary stain in plant cytology.

Sudan black B
p. 98

As myelin stain.

As general fat stain.

As bacterial fat stain.

Bindschedler's
green, p. 102

As oxidation-reduction indicator in study of metabol-
ism.

Toluylene blue
p. 102

As oxidation-reduction indicator.

Indophenol blue
p. 103

By Herxheimer as fat stain.

268

Biological Stains
Table 3. (Continued)

Name of stain
and page
reference*

Application

For chromatin and mucin in general histology; and as
general nuclear stain.

For frozen sections of fresh tissues.

For amyloid (colored blue), mast cells and mucin (red).

Thionin
p. 104

With methyl green and Bordeaux red, by Graberg for
sections of spleen, testis and liver.

By Stoughton for staining bacteria and fungi in plant
tissue; with orange G as counterstain.

End-point staining of Nissl granules.

By Schmorl for staining bone sections.

Azure C
p. 107

By French as nuclear stain; with eosin and orange II.

As nuclear stain; preceding eosin or following phloxine.

In neutral stain combination with eosin; for staining
blood, protozoa, tissue, etc.

Azure A
p. 109

Constituent of MacNeal's tetrachrome stain; for stain-
ing blood.

By Custer for staining bone marrow.

By Kinyoun in a modification of the Ponder diphtheria
stain.

Azure B
p. 110

By Jordan and Heather for Negri bodies.

Important constituent of blood stains, especially Lillie's
modification of Giemsa stain.

A widely used nuclear stain in general histology (zoologi-
cal), with many special applications.

In polychrome methylene blue; as tissue stain.

A favorite bacterial stain, used for many special pur-
poses, as in examination of milk and diagnosis of
diphtheria.

Methylene bhie
p. Ill

By Mallory as nuclear stain, in contrast to phloxine, on
pathological or other histological material.

Staining small animals intra vitam.

Vital staining of nervous tissue.

Tables Relating to Stains
Table 3. (Continued)

269

Name of stain
and page
reference*

Application

Mixed with basic fuchsin; for nervous tissue, in diagnosis
of rabies.

Methylene blue
(Cont.)

For staining blood; in combination with eosin.

With eosin, by Levine as indicator in bacteriological
media, for differentiating colon and aerogenes organ-
isms.

Methylene violet
p. lU

Constituent of MacNeal's tetrachrome blood stain.

•

Counterstain to alizarin red S for calcium deposits.

Methylene green
p. 115

For wood and fixed chromatin, in plant sections. (Does
not stain fresh chromatin.)

By Lison as vital stain for insects.

A metachromatic nuclear stain often useful in place of
thionin or methylene blue. (Pappenheim's panchrome
stain.)

Toluidine bltie
p. 115

Diagnosis of diphtheria. (Albert; Kinyoun)

As general tissue stain.

Histochemical agent for determining thymonucleic acid.

New methylene
blue N, p. 116

By Van Wijhe for cartilage in amphibia.

Brilliant cresyl blue,
p. 117

For blood; to bring out platelets and reticulated cells.

As a vital stain, to indicate 0-R potential.

Staining protozoa and nematodes in animal cytology.

Gallocvanin
p. 119

Nuclear stain for tissue.

Counterstain to protargol, for nervous tissue.

Gallamin blue
p. 119

Nuclear stain; used in form of iron lake.

Celestin blue B
p. 120

Nuclear stain for tissue.

In Lendrum and MacFarlane's modification of Mallory
connective tissue stain.

Lacmoid
p. 120

By Tsvett as microchemical reagent for detection of
callose.

With martins yellow in a stain for pollen tubes.

270

Biological Stains
Table 3. (Continued)

Name of stain
and page
reference*

Application

Lorain Smith's method to distinguish between fatty
acids and neutral fat.

Supravital stain for embryos.

Nile blue sulfate
p. 121

Vital stain for hydrae, protozoa, and yeasts.

For amphibian eggs.

In bacteriological media; to show lipolysis.

Staining sections of bone.

Histochemical studies of Golgi apparatus.

For fixed tissue, (especially of tumors).

Cresyl violet
p. 122

For nervous tissue.

For vital staining of blood.

For fresh tumor tissue.

New blue R
p. 124

Staining fat drops in bacteria.

Resazurin
p. 124

As oxidation-reduction indicator, particularly for milk
tests.

For histological tissue (of embryos); contrast stain to
Janus green.

Nuclei, especially of blood cells, and Nissl granules of
nerve cells, stained intra vitam.

Neutral red
p. 126

"Vital staining" of blood, (i.e., fresh in moist chamber);
also of fresh gonorrhoeal pus.

As indicator in bacteriological culture media; for distin-
gui^ing colon from typhoid organisms, and other
similar purposes.

With light green, by Twort as stain for parasites in tissue.

Histochemical reagent for demonstrating hydrolysis of
fats.

Neutral violet
p. 128

Used in study of chromolysis by Unna.

Pheno-safranin
p. 128

By Moore for colonies of bacteria and fungi.

Tables Relating to Stains
Table 3. (Continued)

271

Name of stain
and page
reference*

Safranin O
p. 129

Amethyst violet
p. 131

Azocarmine G
p. 132

Magdala red
p. 133

Indulin, spirit sol.

p. 134
Indulin, water sol.

p. 134

Nigrosin, water sol.
p. 134

Auramin O
p. 139

Application

A widely used nuclear stain.

For tissues of vascular plants, in combination with
variety of contrast stains. Cutinized, suberized, and
lignified tissue. Spore coats. Protein.

In Flemming's triple stain, for chromatin and other nu-
clear elements, in conjunction with gentian violet;
orange G as contrast stain.

For plant chromatin; with light green or anilin blue W.S.

For bacteria, particularly in contrast to gentian violet
in the Gram technic.

Nuclear dye in certain triple staining procedures.

As tissue stain; especially preceding Mallory's anilin-
blue-orange-G mixture. Heidenhain's "Azan stain."

As counterstain to a resorcin gentian violet.

With iron-hematbxylin and naphthol green B; by MoUier
as tissue stain.

By Kulschitzky for elastic tissue.

By Flemming as nuclear stain.

For negative staining of bacteria.

Some indulin used in counterstaining bulk tissue.

For central nervous tissue; alone or with other stains.

For tissue of pancreas; following hematoxylin.

Pfitzer's picronigrosin for chromatin.

With basic fuchsin in staining bacterial spores (Domer).

By Fischel for vital staining of salamander larvae.

By Vinassa for plant sections.

With cresyl violet; by Kisser as tissue stain.

By Richards and Miller for fluorescent staining of acid-
fast bacteria.

272

Biological Stains
Table 3. (Continued)

Name of stain
and page
reference*

Application

In combination with acid fuchsin and martins yellow;
Pianese's stain for cancer tissue.

For host tissue in plants infected with fungi; used in the
Pianese combination.

By V. Beneden for Ascaris eggs.

Malachite green
p. 141

By Petroff for erythrocytes.

Contrast stain, following borax carmine.

By Shaeffer and Fulton for bacterial spores; with safra-
nin as counterstain.

To replace methyl green in Pappenheim stain.

By Bank as vital stain for epidermis of onion.

Reagent for bacterial polysaccharides (Chapman and
Lieb).

Bacteriostatic or amebicidic agent.

Brilliant green
p. 142

Indicator in bacteriological media for diflFerentiating or-
ganisms of colon, typhoid and dysentary groups
(Krumwiede).

For inhibiting B. coli in stools.

Enrichment of the typhoid organism in broth cultures.

A general plasma stain.

By Benda for spermatozoa; with safranin.

Light green SF

yellowish

p. 143

For cellulose walls in vascular plant tissue; contrast to
safranin.

For general histological tissues; contrast to hematoxylin.

Following safranin; in cytology.

With neutral red; by Twort for parasites in tissues.

Mixed with acid fuchsin, for staining pollen tubes.
(Buchholz).

Fast green FCF
p. 144

As substitute for light green SF yellowish in histology
and cytology.

Tables Relating to Stains
Table 3. (Continued)

273

Name of stain
and page
reference*

Application

A widely used plasma stain in both animal and plant
microtechnic.

Fast green FCF
(Cont.)

With safranin, methyl violet, and orange G, for plant
tissue.

As bacterial stain.

As constituent of Kornhauser's "Quad" stain.

In a modification of the Van Gieson connective tissue
stain.

A powerful nuclear stain; with various green and blue
contrast stains.

For mucin, fuchsinophile granules; for nuclear elements
of central nervous system.

Mixed with methylene blue; for staining Negri bodies in
nerve cells.

Preceding crystal violet; for staining bacilli in tissues.

Basic fuchsin
p. 146

For elastic tissue.

By Williams for Negri bodies.

General bacterial staining, both in smears amd in sec-
tions.

Ziehl-Neelsen technic for staining tubercle organism;
diagnosis by acid-fast property.

In bacteriological culture media for diflFerentiating colon
and typhoid organisms — Endo medimn.

As histochemical reagent for recognition of nucleic acids
and other polysaccharides — the Feulgen stain, HIO4-
Schiff reaction, etc.

A widely used plasma stain.

Acid fuchsin
p. 153

In combination with picric acid; the Van Gieson connec-
tive tissue stain.

In combination with anilin blue and orange G; the Mal-
lory connective tissue stain.

In combination with methyl green and orange G; Ehr-
lich-Biondi-Heidenhain differential stain for sections
and blood smears.

274

Biological Stains
Table 3. (Continued)

Name of stain
and page
reference*

Application

Acid fuchsin
(Cont.)

In combination with methyl green and orange G;
Ehrlich's "Tri-acid stain" for blood smears.

For cortex, pith, cellulose walls, etc., in vascular plants.

Pianese stain for cancer tissue; with malachite green and
martins yellow.

Same technic adapted for staining fungus mycelium in
infected plants.

Bensley-Cowdry stain for mitochondria; with methyl
green.

As Andrade indicator; especially in bacteriological
media.

Hofmann's violet
p. 155

For mast cells — Ehrlich.

For amyloid in sections of tissue.

A powerful nuclear stain.

A very valuable stain in plant histology, particularly for
the achromatic nuclear structures.

Methyl or
Gentian violet
p. 156

For nuclear structures in conjunction with safranin;
orange G as contrast stain — Flemming triple stain.

For fibrin and neuroglia in fresh tissue.

For amyloid; in frozen sections of fresh tissue.

Combined with orange G — Bensley's "neutral gentian",
for demonstrating islands of Langerhans.

In Petroflf's egg medium for cultivation of tubercle or-
ganism.

For bacteriostatic properties.

For staining bacteria in tissues — Gram-Weigert method.

Crystal violet
p. 160

The Gram stain for bacteria; generally with alcoholic dif-
ferentiation to make it selective, and some red or
brown dye as counterstain.

For diphtheria organism and bacterial capsules.

Gram stain for tissues.

Benda's stain for mitochondria; with alizarin red.

Tables Relating to Stains
Table 3. (Continued)

275

Name of stain
and page
reference*

Application

Crystal violet
{Cont.)

Jackson's stain for lightly lignified walls in plants; with
erythrosin.

Following basic fuchsin and picric acid; MacCallum's
stain for influenza bacilli in tissues.

A compound of this dye with bismuth ("bismuth violet")
used for its bacteriostatic properties.

With iodine, in plant cytology.

Ethyl violet
p. 161

In combination with Biebrich scarlet; for staining the
islets of Langerhans — Bowie.

For nervous tissue.

For spirochaetes in blood.

An excellent nuclear stain.

In weak acetic acid solution, for fresh chromatin.

For lignified xylem in plants; with acid fuchsin.

Methvl green
p.' 162

Ethyl green
p. 164

As cytoplasm stain; following acid fuchsin and picric
acid.

In combination with acid fuchsin and orange G; Ehr-
lich's "Tri-acid stain" for blood smears.

In combination with acid fuchsin and orange G; stains
nuclei in sections and in blood — Ehrlich-Biondi-
Heidenhain.

Bensley-Cowdry stain for chromatin; in contrast to acid
fuchsin, which stains the mitochondria.

In combination with pyronin; Pappenheim-Saathof
stain for gonococcus, and for mast cells.

As nuclear stain — Griesbach.

For mucin and amyloid.

For lignified xylem in plant sections; with acid fuchsin.

Iodine green
p. 164

For chromatin in plant tissue; with acid fuchsin for
nucleolus and spindle fibers.

For nervous tissue; with acid fuchsin and picric acid.

For blood; with acid fuchsin.

276

Biological Stains
Table 3. (Continued)

Name of stain
and page
reference*

Application

Iodine green {Cord.)

In a differential medium for the gonococcus.

Spirit blue
p. 166

Counterstain to carmine; especially for embryonic
tissues.

Counterstain to carmine in bulk staining of small ani-
mals and embryos.

Color base used by Knaysi in determining hydrolysis of
fats.

For granules in virus diseases (claimed to be the virus).

Victoria blue 4R
p. 167

As stain for bacterial flagella.

By Lipp as stain for spirochaetes, especially Treponema
pallidum.

A valuable counterstain in animal histology.

Mann's stain for nerve cells; with eosin.

Methyl blue
p. ^^167

Combined with picric acid, as tissue stain; followed by
carmalum or safranin.

For unfixed tissue.

For negative staining of bacteria.

A vital stain for protozoa — Monne.

A good plasma stain, used particularly in nervous tissue
and cartilage.

Anilin blue

water sol.,

p. 168

In Mallory's connective tissue stain; with orange G and
acid fuchsin.

As cytoplasm stain, preceding safranin.

For sections of epithelium; with orcein — Unna.

For nervous tissue.

In bacteriological media for the isolation of the crown
gall organism.

Isamine blue
p. 170

As vital stain by intravenous injection.

Patent blue V
p. 170

For spirochaetes in blood.

For vital staining.

Tables Relating to Stains
Tablie 3. (Continued)

277

Name of stain
and page
reference*

Application

Patent blue (Cord.)

As an acid-base and oxidation-reduction indicator.

As an intracellular indicator.

Xylene cyanol FF
p. 171

In the study of permeability of tissue membranes.

Vital staining of cells in tissue culture.

Rosolic acid
p. 172

With methylene blue in a decolorizing solution following
basic fuchsin; for tubercle organism — Pappenheim.

Victoria blue R
p. 173

For spirochaetes in blood.

Vital stain for yeasts.

Victoria blue B
p. 174

For spermatozoa in testicle.

For nervous tissue.

In form of eosinate, for frozen fresh tissue.

Night blue
p. 174

By Hallberg as stain for acid-fast bacteria.

Wool green S
p. 175

By Lillie as substitute for fast green FCF in the Masson
trichrome stain.

Acridine red 3B
p. 178

With methyl green; by Gomori for staining tissue to
demonstrate calcium despoits.

Pyronin Y
p. 178

In Scudder's combined Gram-Pappenheim stain for
gonorrhoeal pus.

In same procedures as pyronin B.

In combination with methyl green; Pappenheim-Saat-
hof stain for bacteria (especially in organic liquids),
mast cells and other basophile elements.

Pyronin B
p. 180

By Ehrlich in combination with narcein and methyl
green or methylene blue, for blood, etc.

For bacteria; sometimes used as counterstain to gentian
violet in the Gram technic.

By Monn6 for staining mitochondria.

By Huddleson as bacteriostatic agent in distinguishing
species of Brucella.

Rhodamine B
p. 181

With osmic acid; by Griesbach to fix and stain blood
simultaneously.

278

Biological Stains
Table 3. {Condnued)

Name of stain

and page

reference*

Application

As plasma stain, in contrast to methyl green or methy-
lene blue.

Rhodamine B
{Cont.)

By Ehrlich as a component of certain "neutral" stain
mixture; for blood, etc.

Mixed with methylene blue, for glandular tissue —
Houcke.

In fluorescence microscopy.

In microchemical estimation of antimony.

Rhodamine 6G
p. 182

In fluorescence microscopy.

In studies of Golgi apparatus.

Violamine R
p. 182

By Lillie as stain for collagen.

Fast acid blue R
p. 183

By Romell as stain for bacteria in soil.

Fluorescein
p. 186

In fluorescence microscopy.

In culture media for fungi.

Very useful cytoplasm stain.

For algae and fungi; counterstain to hematoxylin, etc.

Eosin Y
p. 187

In combination with methyl blue; Mann stain for sec-
tions of tissues.

By List as cytoplasm stain; preceding methyl green.

For sputum; preceding methylene blue — Teichmfiller.

In combination with methylene blue; for blood smears.

Ethyl eosin
p. 190

Counterstain following Delafield's hematoxylin in
general animal histology.

Stain for Negri bodies in smears or sections of nervous
tissue.

Eosin, bluish
p. 191

Counterstain, especially following Mayer's hemalum.

By Lillie as constituent of neutral stain for tissue, com-
pounded with azure A or toluidine blue 0.

Tables Relating to Stains
Table 3. (Continued)

279

Name of stain

and page
reference*

Application

Substitute for eosin.

Mercurochrome 220
p. 192

Fungicidal agent for treating amphibian embryos.

Counterstain to malachite green in Wirtz spore stain for
bacteria.

Cytoplasm stain for nerve cells; preceding methylene
blue.

Ervthrosin
p. 193

Counterstain for sections of vascular plants; in contrast
to hematoxylin, gentian violet, etc.

Stain for bacteria in dried smears of soil suspensions.

By Jackson as counterstain to crystal violet in plant
histology.

By Gellhorn as vital staining of sea urchin eggs.

For Negri bodies.

For staining algae.

Phloxine
p. 195

Combined with various other acid dyes; used by Unna
in studying chromolysis.

By Mallory as cj'toplasm stain in contrast to methylene
blue, on pathological or other histological material.

Cytoplasm stain, following hematoxylin.

Stain for bacteria, especially those forming slime; also for
dried smears of soil suspensions.

Rose bengal
p. 197

Negative staining of bacteria.

For spirochaetes in blood.

In test for liver function — Delprat and Stowe.

As fluorochrome in study of fats.

Brom cresol purple
p. 204

Hydrogen-ion indicator, used as vital stain.

Brom thymol blue
p. 204

Hydrogen-ion indicator, used as vital stain.

For staining fungi in roots.

280

Biological Stains
Table 3. (Continued)

Name of stain
and page
reference*

Application

Acridine yellow
p. 206

Corphosphine
p. 207

Four dyes used as fluorochromes, for example in insect
histology by Metcalf and Patton.

Acridine orange NO
p. 207

Rivanol
p. 207

Acriflavine
p. 208

With gentian violet; for bacteriostatic properties.

In fluorescence microscopy.

Proflavine
p. 208

For bacteriostatic properties.

Atabrine
p. 209

By Lewis and Goland in vital staining and retardation
of tumors.

In fluorescence microscopy.

Phosphine
p. 209

Following methylene blue; by Schumacher as micro-
chemical reagent for nucleo-proteids.

In fluorescence microscopy.

Rheonine A
p. 210

In fluorescence microscopy.

SaflFron
p. 211

In Masson's trichrom tissue stain.

Staining yellow fever lesions in liver.

As plasma stain in contrast to carmine, either with it or
following it; also with basic fuchsin or other nuclear
stain.

Indigo carmine
p. 212

With acid fuchsin, by Petragnani as stain for