BIOLOGICAL STAINS

A Handbook ox the Nature and Uses of the Dyes
Employed ix the Biological Laboratory.

By H. J. coxx

New York Agricultural Experiment Station
Chairman, CoxfMissiox ox Standardization of Biological Stains

Prepared with the collaboration of:

J. A. Ambler, S. L' Kornhauser, F. B. Mallory, and L. W. Sharp

Members of the Executive Committee
of the Commission

Published by the Commission

Geneva, X'^. Y.
U. S. A.

1925

COPYRIGHT 1925
Commission on Standardization of Biological Stains

COMPOSED AND PRINTED BY
W. F. HUMPHREY,
GENEVA, N. Y., U.S. A

TABLE OF CONTENTS

PAGE

Preface 5

Chapter I. History of staining 7

Chapter II. The general nature of dyes and their classification 11

Chapter III. The spectrophotometric analysis of dyes 25

Chapter IV. Dyes of the nitro, azo, and oxyquinone groups 32

1 . The nitro group 32

2. The azo group 33

3. The oxyquinone group 42

Chapter V. The quinone-imide dyes 44

1. The indamins 44

2. The thiazins 44

3. The oxazins 51

4. The azins 53

a. Amido-azins or eurhodins 53

b. Safranins 55

5. The indulins 57

Chapter VI. The phenyl methane dyes 58

1. Di-phenyl methane derivatives 60

2. Tri-phenyl methane derivatives 60

a. Di-amino tri-phenyl methanes 61

b. Tri-amino tri-phenyl methanes (rosanilins) 62

c. Hydroxy tri-phenyl methanes (rosolic acids) 73

Chapter VII. The xanthene dyes 75

1. The pyronins 75

2. The rhodamines 77

3. Fluorane derivatives 78

4. Phenolphthalein and the sulphonphthaleins 83

Chapter VIII. Compound dyes 86

Chapter IX. The natural dyes 91

The indigo group 91

Cochineal products 92

Orcein and litmus 94

Brazilin and haematoxylin 95

Chapter X. The theory of staining . 98

Appendix I. Tables relating to stains 105

Appendix II. Commission specifications of certain stains 132

Appendix III. Bibliography 138

PREFACE

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 commercial source of supply for them first appeared
in Germany. This was the Grl'ibler Co., later Griibler and Hol-
born. This company did not manufacture the dyes, as used com-
monly 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 sup-
posed 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 manufac-
turers; 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.

Altho a great service was done to biologists by this company in
the latter part of the nineteenth century, such methods of stan-
dardization are not in keeping with modern scientific knowledge.
A recent cooperative undertaking has therefore been organized 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 difiiculties; it is, in brief, difficult to test
each batch before it is put on the market when the concern handling
it is in Europe.

The organization thru 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, altho now no longer a

part of the larger body. It is in effect a coordinating committee
representing the American Chemical Society, the American Society
of Bacteriologists, the Society of American Zoologists, the Botan-
ical Society of America, the American Association of Pathologists
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 chem-
ists, dye manufacturers and stain dealers, so as to be sure that the
needs of biologists can be immediately reflected in the supply of
stains on the market. Its affairs are managed by an executive
committee of five members, the present members of which repre-
sent bacteriology, botany, dye chemistry, pathology, and zoology,
respectively. This executive committee has undertaken the pre-
paration of this book. The authorship of the book has been as-
sumed by the chairman of the committee, however, in order to fix
the responsibility and to make bibliographic 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 assistance 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 in-
formation which has been accumulating in the hands of the Com-
mission since it was organized. It is neither a treatise on dye chem-
istry nor one on microscopy; altho 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 subject 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 men-
tion those of historical significance; but it is realized that there must
be many omissions. It is hoped that readers of the book will co-
operate by calling to the author's attention places where the treat-
ment of any subject seems inadequate.

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

6

CHAPTER I

HISTORY OF STAINING

CONSIDERING how dependent microscopists are today upon
the use of stains, it is hard to reahze that much important
work had been done with the microscope before the use of
stains was attempted. Altho natural dyes such as carmin and
indigo were well known in the early days of the microscope, their
use in staining microscopic preparations does not seem to have been
mentioned until 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 revolutionized
microscopic technic. The early microscopists were able to make
much progress without stains because of their painstaking dili-
gence. The work without stains must have been extremely diflScult,
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 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 — altho of course no one can
deny that modern improvements in the microscope have also
played a part of great importance.

The first use of a dye in microscopic work seems to have been by
Ehrenberg (1838)*, who did not intentionally stain his specimens,
but devised the scheme of grindmg indigo and carmin into a very
fine powder and feeding them to the microorganisms he was study-
ing. His idea in doing this was that the organisms would consume
the dye bodily and that their digestive system could be traced by
observing what portions of the body became colored. Ehrenberg
found by this method that the organisms in question showed cer-
tain zones or bands of color; and assuming that each of these
colored spots was a stomach, he named the group of organisms
"Polygastrica." Inasmuch as the group included bacteria and
protozoa, the name was somewhat of a misnomer. It is interesting,
however, to realize that Ehrenberg's technic is still used to demon-
strate the ability of protozoa to engulf food particles.

The early history of staining from this time on is given in a very
interesting manner by Gierke (1884-5) and by Mann (1902), one

*For references cited see Bibliography pp. 138 to 145.

or the other of which sources is recommended to anyone desiring a
more detailed account than is given here.

Besides the fairly common application of iodine to microscopic
preparations there seems to have been no further effort to color
objects under the microscope until the middle of the century. At
this time carmin was used almost simultaneously by two botanists,
Coppert and Cohn (1849) and by a zoologist Corti (1851). Goppert
and Cohn used the dye to assist them in studying the rotation of
the cell contents of Nitella flexilis. Corti's work is overlooked by
both Gierke and by Mann; but he states definitely (p. 143) that to
observe distinctly the epithelial cells one should color them lightly
with a solution of sugar or of carmin in a mixture of half water and
half alcohol. This work was very promptly followed by another
use of the same dye — the staining of chlorophyl granules in plants
— by Hartig (1854 and 1858). In studying the nucleus of cells he
made use not only of carmin but of litmus and black ink; and he
observed that while albumin and gelatin were easily stained, the
dyes had no action on such material as gums and mucin. Altho
these authors did careful scientific work and certainly were the first
users of dyes for histological purposes, their work apparently at-
tracted no attention at the time. The real introduction of bio-
logical stains was made by Gerlach (1858).

Altho Gerlach did not discover the action of dyes on microscopic
objects, nor was he the first to use carmin, nevertheless he should
be, and generally is, considered the father of the technic of staining.
Having observed that tissues became colored after injection with a
poorly prepared carmin gelatin, he devised the scheme of preparing
an ammoniacal carmin. He was therefore the first to use ammon-
ium carminate, which is so nearly indispensible to modern histolo-
gists. His early efforts with it, however, were unsuccessful — until
he had a lucky accident which revealed the source of his trouble
and opened up the way for further work. He happened to leave a
section of nervous tissue, which had been hardened in potassium
bichromate, over night in a very dilute carmin solution. When he
examined it twenty -four hours later he found that it was beauti-
fully stained, with fine differentiation of nerve fibers and nerve
cells. His earlier failures had been due to the use of too strong a
solution of the dye. This gave the key which helped solve the
problem of tissue staining. Advances came quickly after that; for
Gerlach had shown the way, and others merely had to follow.

These advances were not wholly with carmin; altho at first the
anilin dyes were scarcely kno"v\-n and the number of stains available
to microscopists was quite limited. Indigo was first employed by
Maschke (1859) who was familiar with Hartig's work but not with
Gerlach's. It is stated (anonymous, 1865) that Thiersch and
Miiller had just developed a technic employing carminates in com-
bination with oxalic acid, and a year later Schweigger-Seidel and

8

Dogiel (1866) introduced a combination of carminates with acetic
acid. Haematoxylin was first introduced as an histological stain
by Bohmer (186.5), altho a previous rather unscientific attempt had
been made by Waldeyer (1864) to stain axis-cylinders by means of
the watery extract of logwood. Bohmer's greater success was due
to his use of haematoxylin crystals in combination with alum,
either by accident, or else because he knew that it was frequently
used as a mordant in textile dyeing. Slightly later Frey (1868)
showed that similar results could be obtained by mixing the mor-
dant with the solution in which the tissues were fixed before they
were stained.

Double staining was introduced at about this same period, when
Schwarz (1867) proposed fixing tissue in creosote and acetic acid,
then staining 24 hours in very dilute ammonium carminate, and
subsequently washing and staining for two hours in picric acid. A
year later Ranvier (1868) first used a picro-carmin stain to obtain
the same results by a single procedure.

Anilin dves had become commercial articles before all these ad-
vances with the natural dyes had been made, the first one having
been introduced in 1856 when Perkin prepared mauveine. Fuch-
sin, under the name of anilin red, appeared in 1858. The first sug-
gestion of their use in histology seems to have been made by
Beneke (1862), who used acetic acid colored with a lilac anilin,
probably a mauveine or anilin violet; while two years later Wal-
deyer (1864) used anilin red (fuchsin) and also a blue and a violet
anilin dye. The latter investigator observed the ability of fuch-
sin to stain nuclei more deeply than cytoplasm and the axis cylin-
der more deeply than the medulary sheath of nerves.

The principle of differentiation following staining was soon in-
troduced. Bottcher (1869) differentiated his sections by partially
decolorizing with alcohol after staining with rosanilin nitrate. A
very similar method was later published by Hermann (1875), who
is often mistakenly given the credit for originating the principle.
Later the same procedure was further investigated by Flemming
(1881) who tried both acid and basic dyes, finding that the method
was satisfactory only in the case of the latter group.

Gierke (1. c.) in his historical discussion of staining says that the
history up to his day (1884) was divided into three periods, each
occupying a decade. The first decade, the fifties, was character-
ized 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 carmin 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 depended less upon
chance success by the individual investigator; the effort was made
to use similarlv all the dves 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

9

were available and microscopists were constantly 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 un-
known to the biologist have been shown to be valuable in bringing
out some particular structure; at other times new combinations 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. But the farther this work
has progressed the more the microscopist has become dependent
upon his supply houses to furnish him reliable stains, so carefully
purchased or manufactured that each lot ordered could be counted
upon to duplicate the last.

The preface of this book describes briefly how a company was
formed in Germany to meet the demand for dyes for staining pur-
poses that developed during the last three decades of the nine-
teenth century, and how the recent post-war conditions, together
with the modern demand for a more scientific basis for the industry,
led to the establishment in America of a Commission on Stan-
dardization of Biological Stains. The work of the Commission is
two-fold. First, by cooperation of various biologists and chemists
it is planning to get together all the available information concern-
ing the nature of dyes as related to their use for various purposes in
microscopic technic ; secondly, by working with the manufacturers
it is trying to see that the supply of stains available in America is
of the highest possible qualit}^ as judged by their performance in
actual laboratory use.

The first of these purposes has been partly accomplished by a
series of brief notes appearing in certain biological publications; and
is now being more fully reaHzed by the publication of this book.
The second object is being slowly brought about by the plan of
certifying stains. Manufacturers of stains are being encouraged to
submit to the Commission, for testing, samples of different batches
of their various stains. These samples the Commission compares
by chemical tests and by submitting them to several biologists
skilled in different lines of miscoscopic technic. Then in the case
of those samples which prove satisfactory, the Commission allows
the company to sell the batch tested with a label on it issued by
the Commission, bearing the statement: "Found satisfactory by
Commission on Standardization of Biological Stains for purposes
mentioned on main label. Use for other purposes not contra-
indicated unless specifically so stated on said label." This certi-
fication is issued only for the particular batch tested. The manu-
facturer is furnished only with enough labels to last for that batch
in the ordinary course of trade; and occasional tests of market

10

samples are made to see if any samples sold under the label differ
from the one originally tested. In this way it is hoped before long
to have all the stains on the American market from batches which
have passed chemical tests and have also been tested and approved
by biologists skilled in their use.

When stains are put on the certification basis, specifications for
them, based on the Commission's tests, are prepared and future
lots of stain submitted for certification are in all cases expected to
fulfill these specifications. So far as these specifications have al-
ready been drawn up they are listed in the appendix of this book.

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 logTvood extract (see Chap. IX) are among the most valuable
biological stains; but the natural dyes in general are so few in num-
ber that 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," altho 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.

BENZENE

All coal-tar dyes may be considered as derivatives of the hydro-
carbon, benzene, CeHe, which is the mother substance of the very
important aromatic series of organic compounds. It is an unusual
chemical compound in many respects, and it will be well, in order
to understand the structure of dyestuffs, to review briefly one
theory of its structure which accounts for many of its properties.
The molecule of benzene is composed of six carbon atoms combined
with six hydrogen atoms in such a way that each hydrogen atom is
identical in all its reactions with every other hydrogen atom in the
molecule. Now a carbon atom is considered to have in all cases
four valency bonds, that is it is capable of uniting chemically with

11

four atoms of hydrogen which has a valency of one. The simplest
and best way of expressing these facts by a structural formula is
shown in the figure:

c c

I D

c c

The double bonds in this ring cannot be considered as stationary;
for, if they were, a compound in which two adjacent hydrogen
atoms had been replaced by other elements or radicals should
occur in two different forms according to whether there were a
single or a double bond between the two carbon atoms to which the
substituting elements or groups were attached — which never proves
to be the case. Hence the double bonds must be considered as
mobile, each pair continually oscillating back and forth between
the carbon atom bearing it and the two adjoining carbon atoms.
In practice the formula for benzene is abbreviated to a simple
hexagon :

in which each corner represents a carbon atom. If no chemical
symbol is placed outside the ring at any corner, it is understood
that an atom of hydrogen is attached at that point. This configura-
tion is spoken of as the "benzene ring." When the symbol of some
element or radical is written at a corner, it means that the hydrogen
atom at that point has been replaced by the element or radical to
which the symbol refers.

When two hydrogen atoms are replaced there are only three
possible positions in the molecule which the replacing groups, or
substituents, can take, as shown by the following figures, using
chlorine as the substituent:

CI CI

Cf

CI

In the first formula, the substituents are said to be in the "ortho"
position to each other; in the second they are in the "meta" posi-
tion, and in the third in the "para" position. These three com-
pounds are called respectively: ortho-dichlorobenzene, meta-
dichlorobenzene, and para-dichlorobenzene. The three prefixes are
commonly shortened to the respective initials: "o-" "m-," and

12

**p-." When the compound is complex it is customary to number
the corners of the ring thus :

In naming a compound in this way, the number of the corner to
which a group is attached is given immediately before the name of
the group. Thus, the three compounds shown above may be
called respectively: 1, 2-dichlorobenzene, 1, 3-dichlorobenzene and
1, 4-dichlorobenzene.

There is another type of substitution in the benzene ring which
is very important in dye chemistry. Two atoms or groups having
two valency bonds instead of one may also replace two hydrogen
atoms, provided the replacement takes place simultaneously and
the hydrogen atoms replaced are situated either in the ortho or in
the para position to each other. Thus two oxygen atoms (which
are bivalent) may replace two hydrogen atoms (which are mono-
valent) forming the compound known as quinone CeHA, the
formula for which is

or as commonly written

0

M

C C

U I

c C

0

In printed formulae, such as those that follow in this book, the
quinone ring is often abreviated 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 rear-
ranging 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
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. 84).

Three mono-substitution products of benzene are of importance
in considering the structure of dyes, namely; toluene or methyl-

13

benzene, CeHo- CH3 ; phenol, carbolic acid or phenylic acid,C6H5- OH ;
and anilin or phenyl amine, C6H5NH2. Their constitutional for-
mulae are as follows:

CH, OH NH.

I ■ I I

0 A A

\/ \/ \/

toluene phenol anilin

Two important di-substitution products are xylene or dimethyl
benzene C6H4- (CH3)2, and toluidine, C6H4- CHs- NH2. Both of these
occur in the above mentioned three isomeric forms, as shown below
for xylene :

CH3 CH3 CH3

CH, I 1

\/ \/\ \/

CH3

\

CH,

ortho-xylene meta-xylene para-xylene

CH3

NH

2

ortho-toluidine

CHROMOPHORES, CHROMOGENS, AND AUXOCHROMES

Certain groups of elements are known as chromophores because
when they occur in a benzene derivative they impart to the com-
pound the property of color. The benzene compounds containing
chromophore radicals are known as chromogens. A chromogen,
however, altho it is colored, is not a dye, in that it possesses no
affinity for fibers or tissues. It may coat them, but only mechan-
ically, and it will be easily removed by mechanical processes.
That is, it will not "take." (See, however, the discussion of fat
stains, p. 33). 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 furnish salt-forming properties to the compound.
Certain chromophoric 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

14

of these groups displace three hydrogen atoms in a benzene mole-
cule, we have the compound trinitrobenzene,

0,X NO,

NO.

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 electrolvticallv and consequentlv 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,

OH

O.X I NO,

\/

NO.

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 reduced
to amino groups (-XII2), 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.

Some auxochromes are basic, e.g., the amino group (-XH2),
while others are acidic, e.g., the hydroxyl group (-0H). 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 + O = H— N and H— N +H— CI = H— N

\ / / \ \ / \

H H H OH H H CI

ammonia "water ammonium ammonia Hydro- ammonium

hydroxide chloric chloride"

acid gas

15

H H H H H H H

/ \ \ / / \ /

R— N + O = R— N and R— N +H— CI = R— N

. \ / / \ \ / \

H H H OH H H CI

amine water hypothetical amine hydro- amine

organic am- chloric hy dro-

mon ium base acid gas chloride

The hydroxy! group, on the other hand, is weakly acidic, as it can
furnish hydrogen ions by electrolytic dissociation. The more of
either one of these two 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 hydroxyl 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, altho incapable in itself of conferring
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 extensive elec-
trolytic dissociation . This group, however, is only very feebly auxo-
chromic. 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 introduction 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 what has been said above, it is not to be presumed that
the dyes of commerce are actually bases or acids. Generally the
basic dyes are sold as salts of a colorless acid, such as hydrochloric,
sulfuric, oxalic or acetic acid. Likewise the acid dyes are sold as
their sodium, potassium, calcium or ammonium salts. Occasion-
ally the basic dyes are sold as the free bases, as for example the oil
soluble dyes (see p. 38). When a basic dye which is ordinarily sold
in the form of a salt comes into commerce as the free base, it is
customary to use the word "base" immediately after the name of
the dye. Thus, "basic fuschin" indicates a salt of fuchsin with a
colorless acid, while "fuchsin, base" indicates fuchsin itself, not
combined with an 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

16

into the usual biological stains, and only these need be considered
here. They are as follows :

BASIC CHROMOPHORES

1. The azo group, — X = N — , which is found in all azo dyes, of
which methyl orange 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,

X = X

\/

2. The azin group.

N

/j\

\l/

N

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

H,X X NHa

X

/ \

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

H.X /\ X /\_XH

3. The indamin group, — X = , 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-

17

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.

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 :

C^/~"\^NH

LEUCO COMPOUNDS

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 reduceable, 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 valences 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

18

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:

CH,

_NH.C1+H.0 = H,N_

H,N_/

fuchsin

XH3CI

+0

XH.

leuco-fuchsin

Ordinarily this reaction is reversable under conditions favoring
oxidation. It is of especial significance to the bacteriologist, as
dj^es 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
VI and VII), as soon as the acid radical is removed, the compound
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. 59)
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

\
H.N_/

H.N_/-

C=/

NH.OH

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

H,N

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 manufacturer
as intermediates in the preparation of dyes.

In the case of many acid dyes the chromophore is similarly
broken bj^ a rearrangement of the atoms which occurs on neutrali-

19

zation. This reaction is ordinarily very readily reversable and
makes such dyes useful indicators of acidity. It is discussed more
fully under acid fuchsin (p. 64) and phenolphthalein (p. 83).

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 practi-
cally all colors of the rainbow. It is ordinarily impossible to deter-
mine, a priori, from the chemical formula of a dye what particular
color the compound may have; but there is, nevertheless, a 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 thru red to violets and then greens and blues, as the homo-
logs become higher thru the introduction of successively larger
numbers of methyl or other substituting groups. Thus the com-
pound pararosanilin, which is very frequently sold as basic
fuchsin, but should more properly be called basic rubin, is a tri-
phenyl methane, with an amino group attached to each benzene
ring, but without any methyl groups; thus:

H

\

CI

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

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 thehighesthomologof the series, new fuchsin:

20

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-
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,
Hoffman 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 :

CHj

/

\

/

CH3-N /

\ c

/ \

. / \

CI

\

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 :

1. The nitro dves.

e.g., picric acid.

2. The azo group.

e.g., methyl orange, Bismark broicUy orange G, Congo red,
Sudan III and Sudan IV.

21

3. The oxyquinone group.

e.g., alizarin

4. The quinone-imide group, including

(a) Indamins

(b) Thiazins; e.g., ihionin, toluidine blue, methylene blue.

(c) Oxazins; e.g., brilliant cresyl blue, Xile bine.

(d) Azins, including

(i) Amido-azins; e.g., neutral red.

(ii) Safranins; e.g., safranin 0, inadgala red.

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

5. The phenyl-methane dyes, including

(a) diphenyl-methanes, e.g., auramin.

(b) Diamino tri-phenyl methanes; e.g., malachite green,
brilliant green, light green.

(c) Triamino tri-phenyl methanes; e.g., basic fuchsin, acid
fuchsin, methyl violet, gentian violet, methyl green, anilifi
blue.

(d) Hydroxy tri-phenyl methanes (Rosolic acids); e.g.,
aiirin, corallin red.

6. The xanthene dyes, including

(a) Pyronins; e.g., pyronin G and B.

(b) Rhodamines; e.g., Rhodamine B.

(c) Fluorane derivatives; e.g., eosins, erythrosin, rose
bengal.

(d) Phenolphthalein and the sulphonphthaleins.

DYE NOMEXCLATURE

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-

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
stain Hoffman violet. Then a third laboratory worker may read
both articles and wish to try both methods; so he accordingly

22

orders both dahlia and Hoffman violet. His dealer, who is prob-
ably quite unacquainted with dyes, will very likely send him a
bottle bearing 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 thinking 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 dve mav 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.
106). The preferred designations in this list are the same as in the
earlier, except in the one case of methylene azure. For this stain
Azure I was preferred in the earlier list; but as it was merely a
trade name of somewhat uncertain application methylene azure
seems preferable. The list of synonyms has been revised more
extensively, largely to omit names that are obsolete and have no
present meaning.

DYE INDEXES

Inasmuch as the dye industry originated in Germany and until
the war was almost a monopoly of that country, it is natural that
the first serious efforts to index the dyes should have been under-
taken in that country. Until recently the only important index of
dyes was Schultz's Farbstofftabellen, which is now in its sixth
edition (1923). This index lists all of the important textile dyes,
giving their synonymy, their chemical composition, methods of
preparation, and distinctive characteristics. As these descriptions
are concise, it seemed well to refer to the Schultz number of all the
stains listed in the article on stain nomenclature above mentioned
(Commission, 1923f), wherever such could be given.

More recently another dye index has been published in England
by the Society of Dyers and Colourists (1923). This publication,
known as the Colour Index, is more complete than even the sixth
edition of Schultz, and lists even such dyes as narcein, thionin and
iodine green, which are no longer of use in the textile industry and
have been omitted from recent editions of Schultz. The synonymy
is more complete and up-to-date than that in Schultz, and many
more chemical formulae are given. Accordingly in the following
pages the stains are denoted by their Colour Index number (ab-
breviated C. I. No.) instead of by their Schultz number, as in the

23

list previously published. The Schultz number of each of them is
given for reference purposes, however, in the list in the appendix,
p. 106.

DYE SOLUBILITIES

Textile dyes are never of a high degree of purity. Some of the
impurities are accidental; others are added intentionally so that
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 post-war dyes are much more
pure thail those available before the war. This makes it difficult
to prepare stain solutions identical in strength with those prepared
before the war.

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 dves.

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 cc. 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 on the mar-
ket), it is plain that the two solutions must differ greatly in their
strength. For this reason an early recommendation of the Com-
mission (1923b) was that formulae of the second type be preferred,
on the assumption that a saturated solution of a dye would be more
likely to be of constant dye 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 per-
centage of sodium chloride, for instance, a saturated solution 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 normally.
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, altho possibly more nearly alike than if they had been pre-
pared with identical weights of the dry stain.

As soon as these facts were fully understood, the Commission
(1923e) modified its recommendation. It is plain that the only way

24

two staining 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 used. This can be done only if the
manufacturer has co-operated to the extent of printing the actual
dye content of each batch of stain on the container in which it is
sold. This is not yet commonly done; but the Commission is
issuing its certification only to batches of stain on which the total
dye content is stated. In this way it is hoped that eventually all
stains on the market will be so labeled; and then when staining
formulae are readjusted so as to call for definite quantities of actual
dye, the preparation of staining solutions will be put on a more
scientific basis.

CHAPTER III

THE SPECTROPHOTOMETRIC ANALYSIS OF DYES.

DYES are extremely difficult to analyze by chemical methods.
Their chemistry is in many cases obscure, they often differ
from one another only in the way the chemical groups are
combined together; different dyes may react alike to all known
chemical tests and differ so slightly in solubility that it is difficult
to distinguish one from another. For all these reasons it proves
that spectrophotometric methods offer decided advantages in the
examination of dyes over methods of chemical analysis, both in
respect to general utility and in regard to convenience of appli-
cation.

When a ray of light passes thru a prism it is resolved, as is well
known, into many rays differing from each other in wave length and
in color. Now the color of any substance arises from the selective
absorption or reflection of definite parts of the visible spectrum, as
light passes thru or is reflected from this substance. In the visible
spectrum is included light of wave lengths intermediate between
about 400 and 725 millimicrons. (The millimicron, denoted by the
symbol ijlijl, is one millionth of a millimeter in length.) The color
of the light in the spectrum varies, with increasing wave length,
from violet to red, appearing blue at about 450^ijLt, green at about
500/i^i, yellow at about 550/x/x, and orange at about 600/xju, as shown
in Fig. 1. The color of light which reaches the eye after trans-
mission thru or reflection from a colored substance is comple-
mentary to the color of the light absorbed by that substance. A
violet dye, for example, appears violet because of its predominent
absorption of yellow light. The complementary colors correspond-
ing to the various parts of the spectrum are also shown in Fig. 1
beneath the colors of the spectrum.

The color of substances is ordinarily of complex origin, depending

25

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. Althothe
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.
The essential principle of spectrophotometric analysis may be
understood by reference to Fig. 2, which is a diagram of a spectro-
photometer. Two parallel beams of light of equal intensity enter

Diagram of Spectrum
Showing; complementary colors

Wave lenatk: ^°° <" 500 550 600 650 ^^

*^ ' ' L

> I

Color: violet blue green yellow orange red

Complementary color! yellow orange red violet bh

lue

Fig. 1. Diagram of spectrum

the photometer box by separate orifices, pass thru a prism where
they are resolved into visible spectra, and then reach the eye in
contiguous fields so that very accurate comparison between the
two spectra is possible. The arrangement is such that one beam
passes directly to the prism whereas the intensity of the second
beam may be reduced in any desired proportion by revolving the
photometer circle. , A glass cell containing a dilute solution of the
dye to be exainined is interposed in the path of the first beam and a
similar cell containing water (or whatever solvent is used in the
case of the dye) in the path of the second beam. The spectrum of
the beam which has passed thru 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 spectrum
may be measured by determining the degree to which the intensity
of the light of the second spectrum must be reduced in order to
obtain an equal intensity in the two fields observed by the eye.

,26

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.

irism

divided circle for
pnotometrit adjusW-nt

-wave-length drum
ior rotation of pnsm

tye p.

Diagram of

Spectrophotometer

water cell-

photomaUr Lox

.dy« eeD

Parallel Yearns from
common light source

Fig. 2. Diagramatic section thru a spectrophotometer.

The photometer circle may be calibrated in various ways. In
the general examination of dyes it is convenient to obtain the data
in the terms of a factor known as the Bunsen extinction coefficient
(E), and to employ a circle from which such values may be read
directly.

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. This is done by
setting the drum at some definite wave length and observing
whether both beams of light reaching the eye are of the same in-
tensity; if not, the photometer circle is turned until the beam

27

1^ .

1.0 •

420

460

500

540

580

620

eco

Fig. 3. Absorption curves of five dyes of different colors:

1. Tartrazine (yellow)

2. Orange G.

3. Fuchsin (red)

4. Crystal violet

5. Neptune blue BG.

28

which has not passed thru the dye is of the same intensity as that
which has passed thru the dye cell. A reading of the extinction
coefficient is then made. Further readings may be made at inter-
vals of 10 fjLfjL, with intermediate determinations in the immediate
vicinity of the maximum absorption or at any other point at

IS

1.15

to..

75

.5 '

.25

42.0

4£0

500

540

5&Q

6Z0

ebo ><>*

Fig. 4. Absorption curve of victoria green.

vhich it may appear desirable to bring out detail. If extinction
coefficients are then plotted against wave length a graphic repre-
lentation of the absorption band of the dye is obtained.

If measurements are carried out under suitable standardized
conditions, the spectral position and the general form of the ab-

29

sorption curve are characteristic of the individual dye, while the
magnitude of extinction coefficients (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 indi-
vidual 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.

i.5r

420

4(0

500

Fig. 5. Absorption curves of:

1. Thionin

2. Methylene blue.

3. Apparent mixture of these two dyes, incorrectly marketed (altho in good faith)
as a dye intermediate between them in chemical composition.

The absorption curves of typical yellow, orange, red, violet, and
blue dyes are recorded in Figure 3. It will be noted that their maxi-
mum absorption in each case falls within the range of the comple-
mentary color (cf. Fig. 1). The great majority of dyes of these

30

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 dve. It should never be
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. 4. It has
a principal band in the red 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.

In Fig. 5 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 methylene azure p. 48).
The absorption curve plainly indicates the presence of two dyes,
and suggests their pro})able 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 of
thionin and methylene blue, but occupying an intermediate posi-
tion 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 micro.scopist
or not.

31

CHAPTER IV

DYES OF THE NITRO, AZO, AND OXYQUINONE GROUPS

1. THE NITRO GROUP

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. The best known nitro dye is picric acid.

PICRIC ACID c. I. NO. 7*

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

OH

O.N— r >— NO.

NO.

(An acid dye; absorption maximum about 360 in alcohol)
This compound forms salts by the dissociation of the-OH group,
and the salts have considerable value as stains. Ammonium
picrate is the one most commonly thus used.

Picric acid (or one of its salts) is quite extensively employed in
contrast to acid fuchsin in the VanGieson connective tissue stain.
It is also used as a general cytoplasmic stain in contrast to the
basic dyes. It has further application as a fixative for tissues
that are to be sectioned.!

MARTIUS YELLOW C. I. NO. 9

Synonyms: Manchester yellow, Naphthol yellow.

OH

—NO.

NO.

(An acid dye; absorption maxima about J^}^5, \39d, Sld\)
Martins yellow has been used by Pianese in combination with
malachite green and acid fuchsin for studying cancer tissue; the
same technic was applied to plant tissue by Midler, and is now quite

*This abbreviation stands for the number in the "Colour Index"; see Chapter
II, p. 23.

fFor bibliographic references concerning the procedures referred to in this
chapter see Table 2 in Appendix I, pp. 110-128, and also the bibliography in
Appendix III, p. 138.

32

I

extensively used by plant pathologists in studying sections of
tissue infected by fungi. The dye is also used in preparing certain
light filters used in photomicrography.

AURANTIA C. I. NO. 12

Synonym: Imperial yellow.

This dye is the ammonium salt of hexanitro-diphenylamine.

NO, NO,

\
O.N_/— \_N_

\_/

/ /

NO, NO,

(An acid dye; absorption maximum about J^25)

It is obsolete as a textile dye and is almost unknown as a biological
stain. It is called for, however, in combination with toluidine blue
and acid fuchsin in the Champy-Kull technic for demonstrating
certain cell constituents (mitochondria, etc.)

2. THE AZO GROUP

The azo dyes are characterized by the chromophore — X = X —
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

N = N

The azo chromophore is distinctly basic; but not sufficiently so to
make the dves basic when thev contain hvdroxvl radicals. Those
containing amidogen radicals are, of course, pronouncedly basic.
The position of the hydroxyl or amidogen group on a benzene
ring in relation to the azo groHip is important. Ordinarily they are
in the para position to each other, thus:

N = N

\/ \/\

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

33

compounds. By a rearrangement of the atoms such a compound
is sure to change to a quinoid form, thus :

OH HO

I I il

_N = N_/\ /\_N=.N_/\

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
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
Sudan IV,

N = N CH3

N = N

and

H3C.

are important fat staining dyes.

ORANGE G. C. I. NO. 27

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

S03Na

/\_N = N

I I ;

HO"

SOjXa

(An acid dye; absorption maximum about Jf.85)

This dye is strongly acid because of the two sulphonic groups.
It is one of the most valuable plasma stains in histological work.
It has great use as a background stain for haematoxylin 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 gential 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 Erhlich "triacid mixture,"
also a combination of these same three dyes, is used in staining
blood. A further use is Bensley's "neutral gentian," a combination
of orange G and gential violet for staining the islands of Langer-
hans.

34

BORDEAUX RED C. I. NO. S8

Synonyms: Fast red B or P. Cerasin. Archelline 2B.
Azo-hordeaux . Acid bordeaux.

Various grades denoted: Bordeaux B, BL, G, R extra.

N = N
HO"

NaSO,, SO,Na

{An acid dye; absorption maximum about 520)

Bordeaux red is used as a cytoplasmic stain, in particular when
Heidenhain's haematoxylin is to be used immediately afterward
as a nuclear stain. It has also been used by Graberg with thionin
and methyl green for staining sections, particularly of spleen,
testis, and liver.

JANUS GREEN B. C. I. NO. 133

Synonym: Diazin green.

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 anilin thru an azo group.

CH3
CH3 CH3 /

Y^rVY rYY

/\/ N \/\ /\/ CH3

H.N / \ N = N

CI

(A basic dye; absorption maximum about 592.7)

Janus green is best known for its use in demonstrating chondrio-
somes, stained intra vitam, according to the technic of Michaelis,
and as more recently developed by Cowdry and Bensley. It is
also used by Faris with neutral red for sections of embryos.

FAST YELLOW C. I. NO. l6

{Echt Gelb)

Synonym: Acid yellow.
N = N SOjNa

NaSOj NHa

{An acid dye; absorption maximum about 4.90 in acid solution)

35

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.

METHYL ORANGE C. I. NO. 1 42

Synomyms: Orange III, Helianthin, Gold orange, Trapaeolin D.

N = N CHj

NaS03_| I I i_N

\/ \/ \

CHj

{A weakly acid dye; absorption maximum about 506 in

acid solution)

This dye has little 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 by Bergonzini in the place of orange G in the Ehrlich-
Biondi stain; and byEbbinghaus for staining keratin in sections of
skin.

ORANGE IV. C. I. NO. 1 43

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

NaS03
{An acid dye; absorption maximum about 521 in acid solution)

The onlv biological use of this dve seems to be occasionally as
an indicator.

ORANGE I. Ot^. no. 150

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

N = N

OH

{An acid dye; absorption maximum about ^76)

This is another dye which is turned red by excess of alkali and
has therefore some use as an indicator.

36

ORANGE II. C. I. NO. 151

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

N = N

NaS03 \/\/

(^An acid dye; absorption maximum about Jlt.90)

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.

NARCEIN C. I. NO. 1 52

{An acid dye)

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

AMARANTH C. I. NO. 1 84

Synonyms: Naphthol red. Fast red. Bordeaux. Bordeaux SF.
Victoria rubin. Azo rubin. Wool red.

N = N
/HO \

NaS03 SOiNa

NaSOj

{An acid dye; absorption maximum about 525)

Amaranth is not a commonly used stain, but is of considerable
importance as a food color. It has been used by Griesbach for
staining axis cylinders.

37

SUDAN III. C. I. NO. 248

Synonyms: Sudan G. Tony red. Scarlet G or B. Fettponceau G.

Oil red. Cerasin red.

N=N_

I i HO

N = N"

{A iveakly acid dye; ahsorpiion maximum about 64^1, [590] )

In this dye the hydroxyl group is in the ortho position with
respect to the azo group. As explained above (p. 34), 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 compound
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 coloring fat.
This fact gives Sudan III its chief value to the histologist. It was
introduced as a fat stain by Daddi in 1896.

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.

SUDAN IV c. I. NO. 258

Synonyms: Scarlet red. Scharlach R. Oil red IV.

Fettponceau. Ponceau SB.

CH3

I N = N

H.C

(A weakly acid dye; absorption maximum about 657. 4, [605.5]

in H^SOa)

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

38

position, it has similar physical properties and is fat soluble. It is,
therefore, one of the best fat stains knoTVTi.

BIEBRICH SCARLET, WATER SOLUBLE C. I. NO. 280

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

Double scarlet.

X = X

" r{ X

/\/ I I H0_

xXaS03 /\/

XaSOj N = N

{An acid dye; absorption maximum about 503.5)

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
haematein. It has also been made use of bv Paladino mixed with
alum haematoxylin for double staining effect on histological,
material.

BISMARCK BROWN Y C. I. NO. 33 I

Synonyms: Ve.suvin. Phenylene brown. Manchester brown.
Excelsior brown. Leather brown.

Slightly different shade: Bi-wiarck broivn G.

To be distinguished from :Bismark brown R or GOOO (C. I. No.
332.)

{An acid dye.)

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

XH2

This dye was formerly employed quite extensively as a contrast
stain, but has now been replaced to some extent by others. It is
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 haematoxylin; and occasionally for staining
bacteria in contrast to gential violet in the Gram technic.

39

CONGO RED

C. I. NO. 370

Synonyms : Congo. Cotton red. Direct red.

NHa

/

N = N

X

\.

I NaS03\/\/

NaS03_

\
/

\ /\

N = N

NH.

{An acid dye; absorption maximum about 4-85.)

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) for embryo sections (Schaffer), for staining
plant mucin, and as a general background stain in contrast to
haematoxylin and other nuclear dyes. It has been used by Klebs
as a reagent for cellulose.

TRYPAN RED

C. I. NO. 438

S03Na

NaSOj SO^Na

NaSOi
(An acid dye.)

The chief use of this dye is as a vitstl stain.

SOiNa

C. I. NO. 448 •

BENZOPURPIN 4b

Synonyms: Cotton red J^B. Dianil red 4-C. Diamin red 4^.

Sultan 4^B. Direct red I^B.

NH, NH.

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

CH3

CH3

NaS03 NaS03

{An acid dye; absorption maximum about 497.)

40

This dye has also been used for vital staining; and has been
employed by Zschokke as a plasma stain especially in contrast to
haematoxylin.

TRYPAN BLUE C. I. NO. 477

Synonyms: Chlorazol blue SB. Benzo blue 3B. Dianil blue H3G.

Congo blue 3B. Naphthamine blue 3BX. Benzamine

blue SB. Azidine blue SB. Niagara blue SB.

HaN OH HO NHa

\ I N = N_/~V_/-"\_N = N I /

CH3 CHj /\/\/\

NaSOi SOiNa NaSO^ SOjNa

{An acid dye.)

Apparently the only biological use of this dye is in vital staining.

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

Janus red; C. I. No. <im

Tropaeolin O; C. I. No. 148. Syn: Chrysoin. Gold yellow. Acid
yellow

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

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

Crystal ponceau 6R; C. I. No. 89; Syn: Ponceau 6R.

Carmin naphtha; C. I. No. 24. Syn: Sudan 8. Scharlach B.
Oil yellow.

Alizarin yellow GG; C.I. No. 36. Syn : Anthracene yellow. Ben-
zene yellow.

Chrysoidin R; C. I. No. 21. Syn : Cotton orange. Cerotin orange.

Chrysoidin Y; C. I. No, 20. Syn: Brown salt R. Dark brown
salt R.

Alizarin yellow R; C. I. No. 40. Alizarin orange. Benzene yel-
low PN. Orange R; Anthracene yelloiv RN.

Diamond flavine; C. I. No. 110.

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

Niagara blue 4B; C. I. No. 520. Syn. : Niagara sky blue. Benzoin
sky blue. Dianil blue H 6 G. Congo sky blue. Naphthamine blue.

41

3. THE OXYQUINONE GROUP
The oxyquinone dyes include derivatives of anthracene,

thru its oxidation product anthraquinone

O

O

These dyes are the first to be considered here in which the quinoid
structure occurs. The quinoid ring, which is the most important
chromophore in nearly all the dyes to be discussed in the three fol-
lowing chapters, 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 prop-
erty of combining with metalic oxides to form so-called "lakes",
insoluble compounds of different color from the dye entering into
them. This makes them valuable ones to use after mordanting
with aluminium, iron or chromium compounds.

ALIZARIN

O OH

II / OH

C. I. NO. 1027

II,

O

{An acid dye; absorption maxima about [610.S], 566.5, [5S7.6]

in alkaline solution.)

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. The chief present use of alizarin, however, is
as an indicator.

42

ALIZARIN RED S

Synonyms: Alizarin red, water soluble.

Alizarin sulphate.

c. I. NO. 1034
Alizarin carmin.

SOjNa

{An acid dye.)

This dye, sodium alizarin sulphonate, is used by Benda for stain-
ing chromatin in combination with crystal violet, the chromatin
staining brown, while the mitochondria stain violet. It is also
used as a vital stain for nervous tissue in small invertebrates, and
by Schrotter for sections of nervous tissue.

PURPURIN c. I. NO. 1037

Synonyms: Alizarin No. 6. Alizarinpurpurin.

(An acid dye; absorption maxima about [521.1], Jf85.5,

[Jf.o5.o\ 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.

43

T

CHAPTER V

THE QUINONE-IMIDE DYES

HE dyes of the quinone-imide group contain two chromophore
groups, the indamin group — N = , and the quinoid benzene
ring

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

HN_/"~V=NH

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

/^ N_/— \_NH

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

as:

/\_S _/\=NH

■N=

imido-thio-diphenylimide

In the oxazins, an oxygen atom takes the place of the sulfur of the
thiazins, thus:

\/\_0_/\=NH

oxazin

1. THE INDAMINS

No dye in this group is a common biological stain. The follow-
ing are occasionally mentioned, however, in connection with
histology :

Bindschedler's green. A tetramethyl indamin. C. I. No. 819.

Toluylene blue. A diamido, dimethyl indamin. C. I. No. 820.

2. THE THIAZINS

The thiazins constitute one of the most important groups of
dyes from the standpoint of the biologist; while for textile dying
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.

44

THIONIN C. I. NO. 920

Synonym: Lauth's violet.
(A basic dye; absorption maximum about 602.)*

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
VII). One t>pe is known as the orthoquinoid, the other as the
paraquinoid.

It will be recalled (see p. 13) that when the quinoid ring is formed
the two hydrogen atoms replaced by atoms or groups with 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:

OH

paraquinoid formula
OH

H.X

NH.

"N=

orthoquinoid formula
In the case of the paraquinoid formula the compound is an am-
monium base of the type discussed on p. 15, which is capable of
salt formation thru its pentavalent nitrogen. In the case of the
orthoquinoid formula the salt formation takes place thru 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 preferred.
Possibly both forms actually exist simultaneously. For the sake of
uniformity the paraquinoid form will be sho^-n in the following
pages wherever possible; but with the understanding that the
orthoquinoid form is equally permissable.

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 :

H.X /\ S _/\=XH,

I
CI

"See Fig. 5. p. 30.

It is no longer used as a textile dye, and is very carefully to be
distinguished from thionin blue (C. I. No. 926) which is known to
the trade and is sometimes furnished in place of the desired dye
when thionin is 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 for staining very young bacterial colonies
in his "little plate" technic for coointing bacteria. (Unfortunately
Frost specifies thionin blue in one of his papers, altho the latter
proves entirely unsatisfactory for the purpose.)*

METHYLENE BLUE C. I. NO. 922

Synonym: Swiss blue.

Various grades denoted: Methylene blue BX, B, BG, BB; grade
preferred for biological work: Methylene blue Med. U. S. P.

Methylene blue is a salt of tetramethyl thionin (generally a
chloride, altho other salts are known, such as sulfates). On the
assumption of the paraquinoid structure, it has the formula:

{A basic dye; absorption maximum about 665.)

According to the general rule as to the influence of methylation on
color it is less red in shade than thionin and is therefore a purer
blue. Its absorption curve has a maximum at about 665^tju, with
a lesser peak at about GlO^^t. 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; hence the
meaning of the term Methylene blue Med. U. S. P. The zinc
double salt is less soluble, particularly in alcohol, so for most stain-
ing purposes is less desirable. The investigations of the Commis-
sion show that for all ordinary staining purposes the zinc-free
compound is best; so that is the form at present recommended.

*For bibliographic references concerning the procedures referred to in this chap"
ter, see Table 2 in Appendix I, pp. 110-128, and also the bibliography in Appendix
III, p. 138.

46

Methylene blue is perhaps the stain which the pathologist and
bacteriologist would have the greatest difficulty 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
possibly haematoxylin; and for this reason was the first dye to be
given a thoro 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; secondly, as a bacterial
stain, notably in milk work and in the diagnosis of diphtheria,
where it is especially useful because it has an affinity 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; thirdly 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 other dves like methvlene violet and methvlene azure,
and thus acquire polychrome properties; and lastly as an indicator
in the Levine eosin-methylene-blue medium for differentiating the
colon and aerogenes organisms.

The polychrome properties just mentioned are quite likely to
develop in a methylene blue solution upon standing. Anyone who
has had much experience with the stain is familiar with the oc-
casional green tones from methylene green, the reddish shades of
methylene azure (azure I) and methylene violet. Such a solution
is known as "polychrome methylene blue." Its formation is
hastened by boiling with alkali. In preparing blood stains the
methylene blue solution is treated for this purpose with sodium
carbonate, and then eosin is added, which enters into chemical
combination with the other dyes present, inasmuch as eosin is an
acid dye while methylene blue and its derivatives are basic. The
combination of eosin and methylene blue is often spoken of as the
eosinate of methylene blue. (For a more detailed discussion of the
subject see Chapter VIII.)

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; altho the
recent investigations carried on by the Commission indicate that
the U. S. P. product is sufficiently pure. For blood work there is
frequently recommended 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 specify-
ing 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 a recent paper by Scott and French (IQ'^-tb) it is claimed that
the specially desired staining properties of methylene blue are

47

associated with the presence of lower homologs, particularly the
dimethyl thionins. One of the dimethyl thionins is methylene
azure A; hence the statement of Scott and French is merely another
way of saying that methylene blue should be partially polychromed
in order to have its best staining powers. These lower homologs
are generally present to some extent in methylene blue as sug-
gested by the minor peak in the absorption curve at 610jLl^t. Scott
and French show that a methylene blue may be specially prepared
which contains more than usual of these lower homologs and that
it is better than the ordinary product for their purposes. How
widely their results can be applied to all microscopic uses of methy-
lene blue cannot be decided at present. It is interesting that one
particular instance has come to the attention of the Commission in
which a pure methylene blue was unsatisfactory for a certain
neurological procedure, but a crude textile methylene blue proved
satisfactory. This may possibly have been due to the presence of
other dyes in the impure sample.

One serious bit of confusion has arisen from the designation
^'methylene blue for bacilli" which was used on a certain type of
methylene blue imported before the war. This was the label
placed on a certain type of zinc salt, containing a small amount of
free chloride. Its designation seemed to imply that it was especially
adapted for staining bacteria; recent investigations indicate that
it should rather be considered not good enough for any other pur-
pose! Even for staining bacteria it is not especially satisfactory;
for the most common methylene blue solution of the bacteriologist
is the Loeffler formula, in which a certain amount of saturated al-
coholic solution is used as a stock. Now, since the zinc salt is
nearly insoluble in alcohol, such a stock solution contains little but
the free methylene blue chloride present. For all these reasons the
discontinuing of this grade of methylene blue is decidedly to be
recommended.

METHYLENE AZURE*

Synonyms: Azure I. Azure A. Azure B.

This is one of the components of polychrome methylene blue,
first described by Bernthsen (1885). Definite knowledge of its
chemical nature was gained by Kehrmann (1906) and Bernthsen
(1906). The former showed that there are two azures, the asym-
metric dimethyl thionin. Azure A, and trimethyl thionin, Azure B.
The symmetrical dimethyl thionin, which he prepared, was found
to belong in a quite different category.

*For the following statements in regard to methylene azure and methylene violet
the author is indebted to Dr. W. J. MacNeal.

48

CH,

\

N

CH,

.S.

CH3

CH3

\

H

/

N

/

N H

/ \/\ s

/\

N H

CH3

CI

CI

Tri-methyl thionin
(Azure B, Kehrmann)

Asymmetric di-methyl-thionin
(Azure A, Kehrmann)

CH,

H

\

I

/

N

X^

Symmetrical di-methyl thionin
(not an azure dye)

Bernthsen (1906) and MacNeal (1906) simultaneously and in-
dependently described the easy method of preparing azure by
oxidizing methylene blue by chromate in acid solution and Mac-
Xeal (1925) has perfected this method so that it is now possible to
obtain large yields of Azure A and a fair yield of Azure B.

Azure I (Giemsa) is a trade name applied to a secret prepara-
tion which appears to be a somewhat variable mixture of Azure A
and Azure B. Azure II (Giemsa) is an intentional mixture of
Azure I (Giemsa) with an equal quantity of methylene blue.

The azures are important constituents of all the polychrome
methylene blue stains and are present in undetermined and variable
amounts when these solutions are empirically prepared. For the
preparation of the tetrachrome blood stain of MacXeal (see p. 90),
a definite quantity of Azure A (Asymmetric di-methyl-thionin) is
required.

METHYLENE VIOLET (Bcrnthscn)

Methvlene violet is formed whenever methvlene blue is heated

with a fixed alkali or alkali carbonate. It is a feeble base with the

formula

CH3

N

CH,

.0

"N=

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

49

may be recrystallized from ethylene dichloride (C2H4CI2) 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 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 blood
stain of MacNeal.

Methylene violet (Bernthsen 1885) is not a textile dye and must
not be confused with methylene violet RRA or 3RA, which is C. I.
No. 842.

CH3

CH,

METHYLENE GREEN
NO.

C. I. NO. 924

CH3

N_/\_S _/\ = N-CH,

CI

-N=\/
{A basic dye; absorption maxima at about 660, 607.)

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.

TOLUIDINE BLUE O C. I. NO. 925

Synonym: Methylene blue 0.

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

CH3

\

N

CH,

"N=

,NHaCl
CH.

{A basic dye; absorption maximum about 635.)

Toluidine blue is not ordinarily used for textile dyeing, but is more
easily prepared than thionin — a fact of considerable importance,
as it has properties very much like the latter. It proves, in fact,
that it can be substituted in many ways for thionin, as for example
in staining frozen sections of fresh tissue. It is quite a useful stain,
being an important ingredient of Pappenheim's panchrome stain
for tissues and blood, and also the main constituent of the Albert
stain, which is at present replacing methylene blue in the diagnosis
of diphtheria.

50

NEW METHYLENE BLUE N

Synonym: Methylene blue NN.

c. I. NO. 927

CHi'CHa

\

H

CH3 CH3 CH.CH3

. I I /

N /\_S_/\=N_H

CI

(A basic dye; absorption maxima about [6S6.4], 588.)

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 VanWijhe 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 thru the Commission with samples of various stains to
try, it was 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 methy-
lene blue may have some value in histological work.

3. 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.

BRILLIANT CRESYL BLUE C. I. NO. 877

Synonyms : Cresyl blue 2RN or BBS; brilliant blue C.

CHj'CHa

N

CH.CH

O

.NH,

3 ^AA2

CH.

CI

(.4 basic dye; absorption maxima about 631.8 [579.5])

This dye is prized for certain special work on account of its
highly metachromatic properties. Its chief biological use is for
staining blood to bring out the platelets and the reticulated blood
corpuscles.

Brilliant cresyl blue proved one of the most difficult stains to
obtain in good quality since the war. The problem was finally
solved however, and the pre-war stain has not only been equalled
but surpassed.

51

NILE BLUE SULFATE C. I. NO. 913

Synonym: Nile blue A.

{A basic dye; absorption maxima about 64-^.5, [592.2] )

The use for which this dye is best kno^^'n to the biologist is the
Lorrain Smith fat stain. In this procedure the dye is boiled with
dilute sulfuric acid, and thus hydrolyzed, with the introduction of
oxygen in the place of the radical XHo (S04)k> in other words pro-
ducing 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 combines 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.

Nile blue sulfate is also used unaltered for staining living tad-
poles previous to making transplants, in order to distinguish the
grafts.

CRESYL VIOLET

Synonym: Cresylecht violet (i.e., cresyl fast violet).
{A basic dye; absorption maximum about 585.)

No information is at hand concerning the exact chemical form-
ula of this dye. It is understood to be a derivative of brilliant
cresvl blue.

Cresyl violet is not a widely used stain, but finds some employ-
ment on account of its strongly metachromatic properties. It is
valuable in making permanent preparations of nervous tissue.
According to Ehrlich (1910, II, p. 78) it stains nuclei violet, plasma
blue, amyloid, mucin and mast cell granules red. Williams (19*23)
uses it for staining sections of fresh tumor tissue.

As cresyl violet is not a textile dye, some difficulty has been
found in obtaining it for biological purposes. Williams reports con-
siderable trouble in this respect. Spectrophotometric examination
of the pre-war material used by Williams shows it to have been a
mixture, apparently of cresyl violet with another dye of more red-
dish cast. (See Ambler and Holmes 1924.) The domestic sample
with which he obtained unsatisfactory results on account of its
entire lack of metachromatic properties was this unknown red dye
alone; while the domestic sample with which he obtained good
results, altho not identical with those obtained with the pre-war

52

stain, was true cresyl violet without the reddish dye. True cresyl
violet, like this last mentioned product can now be obtained in
constant quality in America, and proves to have all the properties
needed in this stain.

Other oxazin dyes sometimes mentioned in connection with
histology are:

Capri Blue. C. I. No. 876.

Naphthol Blue. C. I. No. 909. Synonym : Neiv blue B, Fast blue
SR. Phenylene blue. Meldolas blue. Indin blue 2RD.

4. THE AZIXS
The dyes of the azin group are derivatives of phenazin,
C6H4N2Cf,H4, a compound containing two benzene rings linked
thru two nitrogen atoms in such a way as to form a third ring.
Two formula are possible:

N_

"N

and

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, | ,

(see p. 17) is assumed to be the chromophore. The quinoid formula
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 -NHo 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 indicators.
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 becomes pentavalent
and takes part in salt formation.

a.

Amido-azixs 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 indicators.
The best known of the group is toluylene red, base :

CH,

CH3

\
/

N

NH2
_CH3

53

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

NEUTRAL RED

C. I. NO. 825

CH3

CH,

N

{A weakly basic dye.)

CH,

The name of this dye comes from its characteristic neutral color
which is neither red or yellow. It is yellow in solutions a little
below the neutral point (i.e., pH=7.0) in reaction and red in weak
acids, even the reaction 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; altho 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.

As a stain it has special value where a weakly basic, non-toxic
dye is called for. It is used as a vital nuclear stain; 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 is used for bringing out the Nissl
granules in nerve cells; it also has some use in general histological
staining, especially for embriological tissue in combination with
Janus green, as recommended by Faris.

NEUTRAL VIOLET

CH,

\

N_/\_N=/\_NH. -HCl

CH,

N

-NH—

"N^

C. I. NO. 826
CH3

CH3

(A weakly basic dye.)

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
recently used it in a dye mixture employed in the study of chro-
molysis.

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-

54

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 :

N=/\.

H.N / \ NH,

OH

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

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

N_

H,N / \ NH

The commercial safranins are ordinarily methyl or ethyl substitu-
tion products of this; or occasionally phenyl substitution products.
The one of greatest value to the biologist is generally called Safra-
nin O.

SAFRANIN O C. I. NO. 84 1

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

(A basic dye; absorption maximum about 515.)

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

CH3

"N=\/\
H,N / \ NH3 H^ / \ NHa

/\ CI

55

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 515///i.

Safranin O is one of the most important nuclear stains knowTi 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 (see p. 68).

AMETHYST VIOLET C. I. NO. 847

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

/\ N_/\

CH3CH. [ I II CH.CH3

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

N / \ N

/ /\ CI \

CH.CH, I I CH3CH2

{A basic dye; absorption maxima about 589, [5Ji.5.5] )

Amethyst violet has been used by Ehrlich and Lazarus as a basic
dye in certain triple staining technics.

A further dye of this group which the biologist must take into
account, altho it seems to have no significance as a stain, is methy-
lene violet RRA or 3RA, C. I. No. 842 {sy n. : fuchsin or safranin
extra blue.) 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. 49.

MAGDALA RED C. I. NO. 857

Synonyms: Naphthaline red, naphthaline pink, naphthylamine

pink, Sudan red.

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

56

XHa H.X_ I I If NH.

and

(A basic dye; absorption maximum about 521^.)

A true Magdala red put on the market before the war under the
name of Magdala red echt is very expensive. According to Cham-
berlain (19'24 page 58) this is less satisfactory in botanical work
than a cheaper form of Magdala red formerly available not labeled
"echt." Chamberlain further states that he has been able to
obtain recently results with phloxine identical with those which he
used to be able to obtain with the less expensive form of Magdala
red. A sample of the latter examined by the Commission proves
apparently to be erythrosin, — in other words an acid dye of an
entirely different group and very closely related to phloxine. This
makes Dr. Chamberlain's failure to obtain results with Magdala
red echt entirely comprehensible. (See also discussion under
phloxine and erythrosin page 81-8'2.)

Magdala red is used by botanists with anilin blue, in staining
algae. It was used by Flemming as a nuclear stain, and by Kult-
schitzky for staining elastic tissue.

c. THE IXDULIXS

Indulins are similar to safranins but are more complex: being
quite highly phenylated amino derivatives. The only one to
concern us is:

XIGROSIX, WATER SOLUBLE C. I. NO. 865

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

Steel gray. Induliri black.

{A basic dye; absorption maximum about 587.)

The exact constitution of nigrosin is uncertain. It is recom-
mended 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 haematoxylin .
Botanists use it in studying algae and fungi. Pfitzer's picro-
nigrosin serves as a chromatin stain. Nigrosin is also used by
Unna in combination with ''orange" (orange G?) in the study of
the process of chromolysis.

57

CHAPTER VI

THE PHENYL METHANE DYES

ONE of the most important groups of dyes, both from the stand-
point 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 replace
any of the hydrogen atoms with methyl, ethyl, or phenyl groups.
If one H is replaced with CH3, it becomes ethane, CHs-CHs-
If two are replaced with CH3 groups it becomes propane,
'CH3CH2CH3; while if there are three substituent CH3 groups
it becomes iso-butane:

CH, CH,

\ /
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;

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

58

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 :

OH

diphenyl carbinol

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:

NH2

H.N

Now this latter compound contains the necessary auxochrome
groups; but it is not yet a dye. Xo 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.N

NH.^H.N_/~\_C

,NH+H.O

Now this latter compound is the anhydride of a true dye base.
Upon hydration it should theoretically become:

H.N

Such a compound could exist only in watery solution,
only by its salts, the true dyes, as:

It is known

H.N

Altho the theoretical compound given above is the true dye base,
the carbinols are often known as carbinol bases of the phenyl
methane dyes or are sometimes called leuco-bases or color bases.
They are not bases in the chemical sense, however, as they do not

59

have basic properties. As stated above, they lack the chromophore
group, and hence are colorless.

1. DI-PHENYL METHANE DERIVATIVES

The di-phenyl methanes are of practically no biological signif-
icance. Only one deserves mention here.

AURAMIN C. I. NO. 655

Synonyms: Canary yellow. Pyoktaninum aureum.

Pyoktanin yellow.

CH3 CH3

\N_/-\_C_/-\v_N/

/ \_/ I \_/ \

CH3 NHa CH3

CI/

Altho of some use as a drug, auramin has little value in micro-
scopic technic. It has been used by Fischel, however, in the vital
staining of salamander larvae, and by Vinassa for staining plant
sections.*

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

H.N_/~\ H

\_/\ /
C

H.N_/— \/ \/"~\
\_/ \_/

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

H.N /^\ H

H.N /~\/ \/~X NH,

The individual dyes of this series are substitution products of these

*Literature References to the procedures mentioned in this chapter may be
found on pp. 110 to 128 and 138 to 145.

60

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

MALACHITE GREEN C. I. NO. 657

Synonyms: Emerald green. New victoria green. Diamond green.

Solid green. Light green N.

_C

{Absorption maxima: 616, [4^0] )

Malachite green is a rather weakly 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. Today it has
very largely been replaced by methyl green; but it is now often
used by botanists for staining host tissue in plants infected w4th
fungi, according to the technic of Pianese (with acid fuchsin and
martins yellow), which was originally applied to cancer tissue.

BRILLIANT GREEN C. I. NO. 662

Synonyms: Ethyl green. Malachite green G.

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

CH3CH. SO.H / \_N

CH2'CH3

/

CH.CH3

{Absorption maximum: 623.)

The largest call for brilliant green at present is as an indicator
in media for water analysis, according to the technic of Krum-
wiede. It is also used for inhibiting the colon organism in stools;
and is added to broth for the enrichment culture of the typhoid
bacillus.

61

LIGHT GREEN SF, YELLOWISH C. I. NO. 670

Synonyms: Light green 2G, 3G, 40, or 2GN. Acid green (with
various shade designations). Fast acid green N.

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

0x12 'CH^

/
CH3CH2 /^\^N

N_/~\_C I CH2_/^\_S03-Na

/ \_/ l/~\_S03 \_/

NaS03_/'~\_CH, \_/

\_/

(Absorption maximum: 633.5.)

Light green is a valuable plasma stain often used for staining
tissues in contrast to iron haematoxylin, altho it fades badly if
exposed to bright light. It is used by Benda in contrast to safranin
as a cytoplasm stain for spermatozoa. In plant histology it is a
useful cytoplasm and cellulose stain.

b. Tri-amino Tri-phenyl Methanes (Rosanilins) .

The simplest rosanilins are the dyes sold as basic fuchsin. This
term seems to be somewhat loosely used to apply to two or three
different dyes and 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 in-
troduced 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 groups present. There are four primary
compounds theoretically possible, namely with no methyl group,
and with one, two, and three substituent methyl groups respective-
ly. The types commonly encountered are listed below.

Basic fuchsin is a very valuable stain, and is one of the most
powerful nuclear dyes. It is also a stain for mucin, for elastic
tissue, and for bringing out the so-called fuchsinophile granules. It
is often used for staining the nuclear elements of the central ner-
vous tissue. It is one of the most useful bacterial stains, particularly
in the Ziehl-Neelson method for differentiating the tubercle organ-
ism and thus diagnosing tuberculosis. As an indicator it is used to
distinguish the typhoid organism from other closely related forms
by means of the Endo medium, in which it is reduced to the color-
less leuco-fuchsin by the use of sodium sulfite. This medium
(which contains lactose) remains colorless in the presence of the
typhoid organism, which does not attack lactose; but becomes
colored in the presence of organisms like Bacterium coli which fer-

62

ment the lactose (a reaction that converts the leuco-fuchsin again
into the dve fuchsin).

PARA-FUCHSIN C. I. NO. 676

Synonyms: Basic rubin. Pararosanilin. Para-magenta,

H CI

\l

^=

/ "

H

(A basic dye; absorption maximum about 539.)

This dye is frequently sold as basic fuchsin. Investigation, in
fact, shows that most of the stains sold under that name are really
pararosanilin chloride. The acetate is also sometimes encountered.
It is evident that for many purposes for which fuchsin is used, this
is satisfactory; but a recent investigation by the Commission shows
that it is ordinarily less desirable than the higher homologs, es-
pecially rosanilin.

ROSANILIN

This compound is mono-methyl fuchsin, or triamino-tolyl-
diphenyl-methane chloride.

XH2

{A basic dye; absorption maximum about 542.)

It is not a textile dye, and is not found free from pararosanilin
unless specially prepared. One stain company, it is found, has
supplied it as "basic fuchsin, for Endo medium." For this par-
ticular purpose, indeed, rosanilin hydrochloride proves to be the
best adapted of any of the basic fuchsins; and, so far as it has been
investigated, it is a very satisfactory stain for all purposes for
which basic fuchsin is ordinarily used.

BASIC FUCHSIN C. I. NO. 677

Synonyms: Diamond fuchsin. Magenta. Rubin. Anilin red.

Various shades occur, denoted by different designations.

This dye, as furnished for textile purposes, is a mixture in about
equal parts of pararosanilin and rosanilin.

As the greater methyl substitution of the latter compound causes
it to have a deeper shade than pararosanilin, the dye varies in

63

depth according to the amount of rosanilin present. These mix-
tures are the various fuchsins known to commerce. In the course
of the recent investigation of fuchsins, no basic fuchsin of this type
has been found offered to biologists as a stain. Whether it would
prove satisfactory for staining purposes still remains to be deter-
mined.

NEW FUCHSIN C. I. NO. 678

Synonyms: Isonihin. Fuchsin NB.

This compound is tri-methyl fuchsin, or triamino tritolyl

methane chloride:

CH3

H CI /~\_NH.

N_/^_C

_ "\_NH.

H

CH3 !

CH3

{A basic dye; absorption maximum about 5^4, 5J^5.)

This dye is sometimes sold for a stain under the name of basic
fuchsin, altho the most reliable companies sell it under its correct
name. Recent investigations show that it may be admirable for
all the staining purposes for which basic fuchsin is used.

ACID FUCHSIN C. I. NO. 692

Synonyms: Fuchsin 8, S.Y, SS, ST, or S III. Acid magenta.

Acid rubin.

{An acid dye; absorption maximum about 5^5.)

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:

SOj-Na

/
CH3 /— \_NH.

\ IIV_/ /

H2N_/^_C SO3

NaSO,

NH2

64

The bond connecting one of the sulfonic groups with an amino
group attached to a different benzene ring is assumed to exist in
order to account for the fact that altho only two of the sulfonic
groups are neutralized Avith sodium, the compound acts as tho it
has no free acid. In other words, it is a case of intramolecular salt
formation. Now when the tri-sodium salt is formed, this bond is
broken down, whereupon the quinoid ring disappears and the fol-
lowing compound is produced:

SOrXa

H.X

XH.

XaS03 \_/

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 hydrate is called the Andrade indicator. It is used
quite extensively in bacteriological work, because of the striking
reaction when its color is restored by acid-forming bacteria. 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 83 to 86.)

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 connective tissue stain, in which it is used with
picric acid after haematoxylin to differentiate smooth muscle from
connective tissue; the Ehrlich-Biondi stain, in which with methyl
green and orange G it is employed in histology and for staining
blood smears; and the Ehrlich tri-acid stain for blood, which is a
"neutral" combination with orange G and methyl green. In plant
histology it is used to stain the cortex, pith and cellulose walls;
while the Pianese stain (with malachite green and martins yellow),
originally applied to cancer tissue, is now used by plant pathologists
in studying infected vascular plants. It is used with methyl green,
bv Altmann, Benslev and Cowdrv as a stain for mitochondria.
To the pathologist it is quite valuable as a constituent (with anilin
blue and orange G) of the Mallory connective tissue stain.

HOFFMAN VIOLET C. I. NO. 679.

Synonyms : Dahlia. Iodine violet. Red violet. J^iolet R, RR, or J^RN.

Hoffman violet is derived from fuchsin bv the introduction of
methyl or ethyl groups (generally the latter) into the amino groups.
Thus the tri-ethvl rosanilin has the formula:

65

CI

I

H

/

/
"X^C

/

H

\/'

N

' / ■

N

\
H

{A basic dye.)

The dyes known to commerce are mixtures of the higher and
lower homologs of this series. The higher homologs, on account of
the presence of ethyl groups and the higher molecular weight re-
sulting therefrom are a very deep violet.

Hoffman violet has been used by Ehrlich and by Unna for stain-
ing mast cells; and by Juergens for staining amyloid, which stains
red, while the cytoplasm is colored blue.

METHYL VIOLET

C. I. NO. 680

Synonyms: Dahlia B. Paris violet^ Pyoktanin blue.

Gentian violet.

Various shades denoted: Methyl violet 3R, 2R, R, B, 2B, 3B,
BBN, BO, 3V.

The various dyes denoted methyl violet are mixtures of tetra-,
penta-, and hexa-methyl pararosanilin :

H CI

\l

/ "
H

CH,

CH3
C CH,

N

_/ \

CH3

tetra-methyl pararosanilin

CH3 CI

CH,

penta-methy] pararosanilin
66

CH3

/

/ \_

N

CH, CI /\ /

\

\ /

CHj

N / \_C

CHj

/ \ / \

/

CH3 \/^V-

N

\ /

\

CH,

hexa-methyl pararosanilin

(crystal violet)

{Basic dyes; absorption maxima: 583-58 J^ in 90% alcohol.)

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
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
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 crj^stal violet. This
dye, therefore, appeared very interesting to the Commission and
has been given considerable investigation.

CRYSTAL VIOLET C. I. NO. 68 1

Synonyms: Violet C, G, or 7B. Hexamethyl violet. Methyl violet

lOB. Gentian violet.

(A basic dye; absorption maximum about 591.)

This dye is hexa-methyl-pararosanilin, whose formula is given
above as one of the components of methyl violet.

The Commission has made as careful an investigation of this
dye as of any other and has become very enthusiastic over it.
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 triple stain in which it is employed
with orange G and safranin — a technic which gives a very high
degree of differentiation. It is also used for staining amyloid in
frozen sections of fresh and fixed tissue, and for staining the plate-
lets in blood; while it is much used by the Weigert technic for stain-

67

ing 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 bac-
teria. A further more recent use is in bacteriological media for in-
hibiting the growth of Gram-positive organisms, due to its selective
bacteriostatic action.

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 is 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. If crystal violet can be used in all cases, the ad-
vantage 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-
nic crystal violet has been specified instead of gentian violet for
some special procedures. Worth noting is Benda's crystal-violet-
alazirin method for staining chondriosomes, and its modifications
by Meves and Duesberg; and also its use in combination with
erythrosin by botanists for staining lightly lignified walls, in which
technic it proves more uniform than gentian violet.

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, however, and for this reason
the dye is not listed in dye indexes. It apparently applies 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 has been made
and often repeated in biological literature that gentian violet is a
mixture of crvstal and methvl violet; but the looseness of the state-
ment is evident when it is realized that crystal violet is a component
of all the deeper shades of methyl violet. It is possible that before
the war gentian violet did represent a fairly constant mixture, but
there seems to be some doubt even on this point. It is certain that
since the war each company has used its own judgment as to what
to furnish when gentian violet is ordered. One company admits
furnishing crystal violet unmodified under this name; another
claims to be supplying penta-methyl-pararosanilin for gentian
violet; another apparently has been mixing crystal violet and

68

methyl violet 2B together with an equal weight of dextrin. Yet in
actual test, none of these preparations proves any better than
crystal violet or methyl violet 2B, unless it be possibly in inhibit-
ing the growth of Gram-positive bacteria. Unfavorable reports
of crystal violet for this purpose have been received; but tests
of several samples of crystal violet now on the market show
them to be bacteriostatic to a higher degree than the sample of
pre-war gentian violet with which they were compared.

Under the circumstances the Commission has faced a difficult
problem in trying to standardize gentian violet. The question has
been whether to recognize the name at all, or to approve some par-
ticular 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 gentian violet under that name. The second
course (unless considerable latitude be recognized) would be en-
tirely arbitrary, inasmuch as no information is available to show
which members of this group of dyes are especially needed in hist-
ology or bacteriology. Accordingly the Commission has finally
defined gentian violet as either penta-methyl or hexa-methyl para-
rosanilin, or else a mixture of methylated pararosanilins composed
primarily of the two compounds just named and having a shade at
least as deep as that recognized in the trade as methyl violet 2B.
Gentian violet, must, moreover, prove satisfactory in actual use
as determined by the tests proposed by the Commission (see
specifications page 135). This definition will not exclude anything
now sold as gentian violet except those that do not prove satis-
factory in performance. At the same time the Commission does
not wish to give official standing to gentian violet and recommends
that crystal violet be ordinarily ordered in its place.

METHYL GREEN C. I. NO. 684

Synonyms: Double green. Light green.
{A basic dye; absorption maximum about 633.8)

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:*

*This ordinarily occurs in trade as a zinc double salt.

69

As the seventh methyl group is very loosely attached, there is
always some methyl violet present, either because it is not all com-
pletely 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 be always
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. In the Ehrlich-Biondi technic it is used to stain nuclei in
contrast to acid fuchsin; while Bensley employs it to stain chroma-
tin in contrast to acid fuchsin which stains the mitochondria. It is
an ingredient of the Ehrlich triacid mixture (with orange G and
acid fuchsin) for staining blood smears. Botanists find it a valu-
able stain, combined with acid fuchsin, for lignified xylem. One of
its most valuable uses today is in the Pappenheim 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 material 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 completelj^ 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 ordinarilv 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. That this is not generally
realized is shown by the fact that when a certain company recently
announced for sale a supply of German stains imported before the
war and kept on their shelves since then, one laboratory ordered a

70

pound of methyl green. For the purposes for which this stain was
desired this batch may be satisfactory; but the user can hardly
count on its being the same as it was when imported nor on its
remaining unchanged until used up.

IODINE GREEN

Synonym: Hoffmann s green.

c. I. NO. 686

This dye is closely related to methyl green, the generally accept-
ed formula being:

CHj CI

\l
N_

/ "
CH,

CI CHj

1/
/-\_N-CH3

/V_/ \
/ CH3

.=C CHj

./ \ ^ /

\ CH3

CHj

{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
is used by Ciaccio for nervous tissue in combination with acid
fuchsin and picric acid; and by Lefas as a blood stain in combina-
tion with acid fuchsin. It is used by Zimmermann with basic
fuchsin for staining chromatin in plant tissue; while together with
acid fuchsin it is occasionally used by botanists for staining lignified
xylem. It is also used for staining mucin and amyloid, having the
property of giving the latter a red instead of a green color.

SPIRIT BLUE

C. I. NO. 689

Synonyms: Gentian blue. Anilin blue, alcohol soluble. Night blue.

Lyons blue. Paris blue.

This is a mixture of di-phenyl rosanilin

CH,

71

and tri-phenyl rosanilin:

CI

I

N_

T

H

_C

/

H

I

N

\_/

{Basic dyes; absorption maximum of spirit blue 2R
about 581 in alcohol.)

It is used in contrast to carmin in staining embryonic tissues; it
brings out growing nerve fibers well.

METHYL BLUE C. I. NO. 706

Synonyms: Cotton blue. Helvetia blue.

NaSO,

H

I

N

SO.Na

{An acid dye; absorption maximum about 607.)

On account of the sulphonic groups, this dye is strongly acidic
and makes a good counter-stain. It is used by Mann with eosin
for staining nerve cells; and by Dubreuil, combined w4th picric
acid, in contrast to a red nuclear stain such as carmin or safranin.

ANiLiN BLUE, w. s. (i.e.. Water soluble) c. i. no. 707

Synonyms: China blue. Soluble blue 3M or 2R. Marine blue.
Cotton blue. Water blue. Berlin blue.

This dye is a mixture of the tri-sulphonates* of tri-phenyl para-
rosanilin (C. I. 706) and of di-phenyl rosanilin. The latter is:

•"The location of the sulphonic groups is uncertain.

72

NaSO.,

ch/

SO,Xa

(An acid dye; absorption maximum of water blue 2B

about 5Jf.6.5.)

It is a widely used histological stain, having valuable counter-
staining properties. It is also of use as an indicator, due to the dis-
appearance of the color upon complete neutralization, as in the
case of acid fuchsin. As an indicator, however, it has the disad-
vantage that the blue color is but slowly restored upon addition of
acid.

Its chief histologica uses are : by Stroebe and Huber as a cyto-
plasm stain preceding safranin; by Galli for axis cylinders; fre-
quently by botanists as a contrast for safranin in vascular plant
tissue, or for magdala red in algae; and very widely by pathologists
in the Mallory connective tissue stain, in which it is combined with
orange G and acid fuchsin; by Unna in contrast to orcein for stain-
ing epithelial sections, and in studying the process of chromolj^sis.

(c) Hydroxy 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
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,
since in acid solution the quinoid ring disappears and the compound
becomes colorless, while alkali changes it back to the colored form.
Thus:

H /—\ OH

HO

OH

+2XaOH =

leuco-rosolic acid

+ sodium =
hydroxide

_C

+2H.0

disodium rosolate

7S

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 :

H.N /~\ /^ NH. HO /^\ /—\ OH

OH

.NH^
HO

Pararosanilin carbinol

leuco-pararosolic acid

Rosolic acid, on the other hand, is a mono-methyl derivative, and
bears the same relation to rosanilin :

H.N

CH,

NH.

HO

OH

c / c

/ \/~\_NH2 CH

HO \_/

Rosanilin carbinol leuco-rosolic acid

Now the dye to which the name rosolic acid or aurin is generally
given in practice is a mixture consisting of both rosolic acid and
pararosolic acid together with other closely related compounds.
This dye is:

AURIN OR ROSOLIC ACID C. I. NO. 724

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.

Corallin yellow is the name given to its sodium salt.

No other dyes of this group have biological use. Two others
perhaps deserve mention:

CORALLIN RED C. I. NO. 726

Synonym: Aurin R.

Apparently a compound dye, the pararosanilin salt of pararosolic
acid.

CHROM VIOLET

A carboxyl derivative of pararosolic acid :

COONa

0_/~X—

C. I. NO. 727

/
COONa

COONa

74

CHAPTER VII

THE XANTHENE DYES

THE group of colored compounds known as xanthese 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 compound

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

/
H,N_/\_0_/\_N— H

CI

H.N

or

NH.

Another arrangement of the atoms is possible in which no quinoid
ring exists, namely:

H.N

CI

I

o

NH.

"C"

I
H

This latter form might also be assumed for the oxazins and thiazins
as well, and this type or 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

75

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 admissable; and it is
possible that the compound occurs in two or even all three of the
different forms.

PYROXIN G

c. I. NO. 739

CH,

CH,

\

I

/

N

CH3

/
_X— CH3

"I
CI

{A basic dye; absorption maximum about 5^5.)

This dye finds its principal use as a stain in the Pappenheim
combination, where it is employed with methyl green for staining
basophile elements, especialty the mast cells, and for staining the
gonococcus in smears of pus. It is also used sometimes as a counter
stain in the Gram technic for bacteria; and by Ehrlich as a com-
ponent of certain "neutral" stains. Since the war it has proved
difficult to obtain this dye in America except by importation, as
the intermediate from which it is manufactured is very difficult to
prepare and is not produced in this country.*

CH.CH

3 ^^^2

\

N

PYRONIX B C. I. NO. 741

CH.CH3
O /\_N— CH. •CH3

CH.CH

3 '-'^^2

{A basic dye; absorption maximum about 550.)

This dye differs from pyronin G 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.
Investigations recently carried on by the Commission indicate that
it can replace pyronin G in the Pappenheim stain and probably in
all its other uses. This is very encouraging, for it is much more
easily prepared and a very satisfactory product of American
manufacture is now available.

*For literature references to the procedures mentioned in this chapter see
pp 110 to 128 and 138 to 145.

76

2. THE RHOD AMINES

The rhodamines are similar to the pyronins except that there is
a third benzene ring attached to the central carbon atom and
attached to this ring is a carboxyl group in the ortho position.
This latter group, altho of acid tendency, does not counteract the
basic action of the amino groups, so the dyes are basic in character.
Only one of them is of any significance to the biologist, namely:

CH.CH

3 ^^^2

RHODAMINE B C. I. NO. 749

Synonyms: Rhodamine 0. Brilliant pink.

CH2CH3

O /\_N— CH.CH

"I
CI

GH3'CH2

I

COOH

{A basic dye; absorption maxima about 556.5^ [519])

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; by Rosen
for histological work in contrast to methylene blue; and by others
in contrast to methyl green.

A somewhat different dye, knoTvn 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 CgH^COOH being replaced by C2H4COOH.

In practice the rhodamines are prepared not from xanthene but
by the condensation of two molecules of meta-amino phenol,

NH.

OH

with one of phthalic anhydride :

This shows their close relation to the next group of dyes, namely
the fluorane 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.

3. FLUORANE DERIVATIVES

Fluorane is not a dye, but is a very important compound in dye

chemistry. It is a derivative of phthalic anhydride, 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

c

I "CO

The fluorane 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.

FLUORESCEIN C. I. NO. 7 66

Synonym: Uranin.

This is the simplest of the fluorane dyes, and is the mother sub-
stance of the eosins. The composition of its sodium salt is:

NaO

O /\-0

'C=

COONa

\/

(An acid dye; absorption maximum about 4-90.)

78

EOsiN, Y (i.e., yellowish) C. I. NO. 768

Synonyms: Water soluble eosin. Eosin W or WS.

Various shades denoted: Eosin G, Y extra, S extra, J extra,
B extra, GGF, 3J, 4J, KS, DH and JJF.

{An acid dye; absorption maximum about 516.)

This dye is typically tetrabrom fluorescein :

Br Br

NaO

COONa

but the mono- and di-brom derivatives are also known and fre-
quently occur in eosin. This affects the shade, as the more bromine
present the redder the dye. It is plain that various mixtures of
these compounds are on the market; but it has not yet been deter-
mined which are more suitable for biological purposes. Consider-
ably more work on eosin is needed than has been done at the
present time. From the name "water soluble eosin" it is often
assumed that this dye is not soluble in alcohol. This is not true,
however.

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 thru the use of this dye.
It is often employed as a counterstain for haematoxylin and the
green or blue basic dyes; as for example by Mallory with methy-
lene blue,* and by List with methyl green. It is used by Mann
mixed with methylene blue as a tissue stain; and by Teichmuller
for staining sputum before staining with methylene blue. 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.

METHYL EOSIN C. I. NO. 769

Synonym: Eosin, alcohol soluble.

This is the methyl ester of yellowish eosin, the sodium salt of
which is:

*See, however, the statement about this technic below under phloxine (p. 82)

79

Br

Br

NaO

\/\. « A-«

Br

Br

COOCH3

(An acid dye; absorption maxima about 520, [It.85.5] )

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

I

NaO

Br

Br

"COOCH.CH3

{An acid dye; absorption maxima about 523.5, [Ji-87] )

An alcohol soluble eosin, either this dye or the preceding, is a
valuable counterstain after Delafield's haematoxylin in general
animal histology.

EOSIN, BLUISH

C. I. NO. 771

Synonyms: Eosin BN, J5, BW, or DKV. Saf rosin, Eosin scarlet
B or BB. Scarlet J, J J, V. Nopalin G. Caesar red.

This is a dibrom derivative of dinitro-fluorescein.

Br Br

NaO I I

O /\_0

NO2

NO,
"COONa

{An acid dye; absorption maxima about 521.5, {Ji-86\ )

80

ERYTHROSIN, YELLOWISH

C. I. NO. 772

Synonyms: Erythrosin R or G. Pyrosin J. Dianthine G. lodo-

eosin G.

This is a fluorescein in which there are two substituent iodine
atoms instead of four bromine atoms as in yellowish eosin.

O

_0

I
COONa

{An acid dye; absorption maximum about 510.5.)

ERYTHROSIN, BLUISH

C. I. NO. 773

Synonyms: Erythrosin B. Pyrosin B. Eosin J. lodo-eosin B.

Dianthin B.

This is the tetraiodo compound corresponding to the tetrabrom
compound of typical eosin.

I I

NaO I I

O /\_0

COONa

{An acid dye; absorption maximum about 524--)

Erythrosin has some use as an indicator. It is also em-
ployed as a contrast stain for haematoxylin and certain blue and
violet nuclear stains. Held uses it, preceding methylene blue, as
a plasma stain for nerve cells. It is employed by Winogradsky for
staining bacteria in soil. For these purposes probably the tetra-
iodo compound (i.e., erythrosin bluish) is desired; but the litera-
ture 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 57) 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.

81

PHLOXINE C. I. NO. 7 74 OF 778

Synonyms: Erythrosin BB. New pink.

(An acid dye; absorption maximum of C. I. No. 778 about 537.5.)

If fluorescein is prepared from dichlor or tetrachlor-phthalic
acid, instead of from simple phthalic acid, its derivatives have a
deeper, more pleasing shade than the ordinary eosins. Of these
derivatives, phloxine contains four bromine atoms and thus cor-
responds to Eosin Y. It contains two or four chlorine atoms ac-
cording to whether it is prepared from the dichlor- or the tetra-
chlor-phthalic acid; and there are two Colour Index numbers cor-
responding to these two compounds, the former being No. 774, the
latter No. 778. It is uncertain which is desired by the biologist.

The dichlor compound is :

Br Br

NaO I I

O /\_0

Br I Br

Cl_/\_

COOXa

CI

Unna uses phloxine in combination with several other acid dyes
in studying the process of chromolysis. The dye has seldom been
specified for biological work; yet there is reason to believe that it is
a more valuable stain than anyone has realized in the past, and that
it has frequently been used under other names.

Chamberlain (19'24 page 58) 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.

It has, furthermore, been found that no sample of eosin at
present available of either domestic or foreign origin works in the
well-known Mallory eosin-methylene-blue stain ; but after investi-
gating both phloxine and rose bengal. Dr. Mallory reports the
former to be "the best eosin I have yet found for use in the
eosin-methylene blue stain for parafin 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 incor-
rect name used in the publication of a well-known technic.

*Quoted from personal letter.

82

ROSE BENGAL C. I. NO. 777 OF 779

Various shades denoted: Rose bengal B, 2B, 3B.

{An acid dye; absorption maximum of C. I. No. 779 about 5^8.)

This is a second derivative of di-chlor or tetra-chlor fluorescein,
and corresponds to erythrosin, as it contains four iodine atoms. As
in the case of phloxine, there are two similar dyes, the di-chlor
compound (So. Ill) and the tetra-chlor compound (Xo. 779); it
is not certain which is desired in microscopic work. The former
has the formula

NaO

O

C

_0

I I

ci_/\_

COONa

•CI

This dye has a pleasing deep pink color; and altho an acid dye
it proves to have considerable affinity for bacterial protoplasm,
if used in carbolic acid solution, and to have good selective prop-
erties when used as a bacterial stain. It has recently been recom-
mended by the author for staining bacteria in soil suspensions. It
has also been used as a cytoplasm stain following haematoxylin.

4. PHEXOLPHTHALEIN AND THE SULPHOXPHTHALEINS
A phthalein is a compound of phthalic acid :

COOH

I

COOH

or rather of phthalic anhydride:

CO

CO

o

with phenol or a phenol derivative. If phthalic acid is heated
with phenol and sulfuric acid it combines with two molecules of
the latter and forms phenolphthalein. In the same way, a sul-
phonphthalein is a compound of ortho-sulpho-benzoic acid:

SO3H

COOH

83

and phenol or a phenol derivative. These compounds, altho some-
times behaving as dyes, are not used as dyes or stains, but as in-
dicators. For this purpose the members of the group are very
valuable.

Phenolphthalein, altho 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

o

I I c = o

\/

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:

H0_/\ /\_0

I l_ =1 I
\/ C \/

_ COOXa

\/

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.

The sulphonphthaleins are not commercial dyes, altho capable
of acting as acid dyes. Their real value is as indicators. 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. Fortunatelv nearlv everv one of them has a different

*/ t t-'

point in the hydrogen-ion scale at which its color change begins, so
that between them they indicate very accurately the hydrogen-
ion concentration of solutions of any reaction ordinarily encoun-
tered. Some of them, such as thymol-sulphonphthalein (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 disap-
pearance and reappearance of the quinoid ring, is generally as-
sumed; but in the case of these compounds the relation of structure
to color is complicated and has not yet been worked out to general
satisfaction.

84

Phenol-sulphonphthalein {^phenol red) has the formula :

HO OH

\/\ /\/

c_
/ o

so,

\/

Among the other important sulphonphthalein indicators: Brom
cresol purple is di-brom-ortho-cresol-sulphonphthalein:

CH3 CH3

HO I I OH

Br C_ Br

I O

"SO2

Thymol blue and Bromthymol blue are thymol-sulphonphthalein
and di-brom-thymol-sulphonphthalein respectively; thus:

CHj CH3 CH3 CH, CH3 CH3 CH3 CH

3

CH CH CH CH

HO I I OH HO I I OH

. '\ /\/ \/\ /\/

11 II and 11

I Br I \ / I Br

CH3 C_ CH3 CH3 C_ CH3

/ o / O

I I so, \ \ so.

The formulae are all so similar that there is no need of giving the
others. It is enough to state that brom phenol blue is tetra-brom-
phenol-sulphonphthalein ; cresol red is ortho-cresol-sulphonphtha-
lein ; brom cresol green is tetra-brom-meta-cresol-sulphonphthalein ;
chlor phenol red is di-chlor-phenol-sulphonphthalein; brom phenol
red is di-brom-phenol-sulphonphthalein; while brom-chlor phenol
blue is di-brom-di-chlor-phenol-sulphonphthalein.

85

CHAPTER VIII
COMPOUND DYES

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 with 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, and the resulting solution, when it reaches equi-
librium, 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 substi-
tuted for ammonium nitrate, it is thrown out of solution and equi-
librium is not reached until the solution is free (or at least practi-
cally free) from the two ions which are insoluble in combination.
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 respectively; 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 thro\\Ti out of solution;
hence the compound dye can be formed in considerable quantitj'^.

Now compound dyes of this sort are sometimes referred to as
neutral dyes or neutral stains. This terminology, of course, does

86

not indicate that they are neutral in reaction any more than do the
corresponding terms acid and basic dyes. A dye chemist, in fact,
uses the term neutral dye in an entirely different sense; but it is
frequently employed by biologists, especially by Ehrlich, to refer
to the basic dye salts of dye acids. In this chapter these compound
dyes will be called neutral stains, as this latter term is not employed
by dyers or dye chemists in any sense, and is unlikely, therefore, to
be misunderstood. Erhlich also uses another term to apply to
some of these compounds, namely "tri-acid dyes." He uses this
term on the assumption that, while in the ordinary basic dyes only
one of the three affinities of the dye for acid is satisfied, it is pos-
sible to satisfy all three and in this wav to saturate the basic dve

«. »-■ t,

with acid. This assumption of his seems to be incorrect; but
Ehrlich's "tri-acid stain" (see below) is so well-known that an
explanation of the term is necessary.

It is possible to obtain endless variety of such dyes; but in prac-
tice only a certain number of them have proved useful. Among
the basic dyes the most suitable for this purpose are methylene
blue and the rosanilins (which act as strong ammonium bases);
among the acid dyes eosin and the sulphonic acids (e.g., orange G
and acid fuchsin).

Altho the neutral stains are insoluble in water, they are soluble
to a greater or less extent in excess of either the acid or the basic
dye. Thus if a watery solution of acid fuchsin is neutralized by
adding drop by drop a watery 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
dye is precipitated and the solution becomes nearly colorless. Then
if more methyl green is added the neutral dye is slowly dissolved
again; but as a rule neutral dyes are less soluble in excess of base
than in excess of acid.

As simple aqueous solutions of these dyes are impossible 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 dye in solution ; sometimes (as
in the original Romanovsky stain) the compound dye is used im-
mediately after mixing, before the reaction is complete or pre-
cipitation 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 mav be diluted with water.
This latter method is particularly efficacious, because the disso-
ciation which takes place upon the addition of water causes the
production of various dye compounds which may stain intensively
and very selectively.

87

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"Triacid Stain."

The first neutral stain proposed for microscopic work was the
"triacid stain" (seeEhrlich 1910, 1, 227). In forming this compound
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 dve. This excess of the acid dve allows
the neutral stain to stav in solution. The dve thus formed is a
very valuable blood stain, and brings out finely the different struc-
tures in the leucocvtes.

Slight modifications of this triacid stain have been used for
tissues. The best known of these modifications is that of Biondi-
Heidenhain.

Eosin-Methylene-blue Compounds*

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 ex-
cellent for blood, particularly in bringing out the malarial parasite.
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, alt ho it was subsequently
named azure I or methylene azure; its true chemistry has scarcely
been understood until very recently (see p. 48). Methylene violet,
which probably was also present, had already been described by
Bernthsen (1885) . How these new dyes were formed in the Roman-
ovsky stain was not known then; altho 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.

Present day blood stains are often spoken of as modified Roman-
ovsky stains; altho 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

*A good account of the history of these blood stains is given by MacNeal (1906).

88

to the formation of other dyes by the decomposition of methylene
blue. Unna (1891) had already described what he called poly-
chrome methylene blue, made by heating a solution of methylene
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 properly neu-
tralized 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 neutral-
ization.

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;
but it was an important step in that he showed the possibility of
collecting the precipitated compound stain and of dissolving it in
some solvent other than water. Combining this procedure with
the Nocht stain was the next logical step and was taken inde-
pendently by Renter (1901) and by Leishman (1901). The method
thus introduced was briefly to follow Nocht's technic of combining
eosin with polychrome methylene blue, but then to filter off the
precipitate and to redissolve it in methyl alcohol, not adding fur-
ther 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 modifications, the one most used
in America, (see Mallory and AYright, 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 Leish-
man technic calls for twelve hours at 65°, with subsequent stand-
ing for ten days. Balch's modification calls for a polychrome
methylene blue prepared by standing ten days with precipitated
silver oxide.

Giemsa's and MacNeal's modifications are somewhat different.
Giemsa obtained methylene azure, in what he considered a pure
form, and combined it with eosin in order to obtain a more definite
compound than when polychrome methylene blue is used. Then
to obtain better differentiation he mixed it with methylene blue.
Following his instructions, the Griibler Co. put on the market a
product known as Azure II, which was a mixture of Azure I (i.e.,
methylene azure) and methylene blue in equal parts; and also a
compound known as Azure Il-eosin, which was an eosinate of
Azure II, or more precisely a mixture of the eosinate of methylene
blue with that of Azure I, in equal parts. This latter compound is
the one generally known as the Giemsa stain. MacNeal (1922)
proposed a method for obtaining a very similar blood stain, pre-

89

pared on even more scientific principles. This stain, known as the
tetrachrome blood stain, is prepared by mixing definite proportions
of methylene blue, methylene violet, methylene azure, and eosin.
When first proposed (1922) this stain was to be prepared with a
crude methylene azure, the pure product being at that time diffi-
cult to prepare and therefore expensive. MacNeal's latest work,
however (1925), shows a simple method of preparing methylene
azure A (asymmetric di-methyl-thionin), which is apparently the
important ingredient of Azure I, and he now specifies azure A in
the tetrachrome stain instead of the less definite product methylene
azure, as formerly. Azure A for this purpose can already be ob-
tained on the market in America.

Some difficulty was experienced at first in compounding the
eosin-methylene-blue blood stains when imported dyes were no
longer available. In some cases these difficulties were probably
due to poor methylene blue or to poor eosin; but upon investiga-
tion the solvent, rather than the dyes themselves, has been found
to be most often at fault. As stated above, the precipitated com-
pound dye must be dissolved in methyl alcohol; but there are many
grades of methyl alcohol and not all are equally suitable for the
purpose. Apparently absolute purity is not needed; but two points
are very important; the methyl alcohol must be neutral in reac-
tion, and it must be free from acetone. In specifying a methyl
alcohol for the blood stains, these two properties should be in-
sisted upon. Very good methyl alcohol for this special purpose is
now on the market.

Other Compound Stains

Various other compounds of acid and basic dyes have been used
for special purposes. The basic dye employed in these compounds
is ordinarily methyl green or methylene blue; but sometimes basic
fuchsin, pyronin or rhodamine is used. The most commonly used
acid dyes are eosin, orange G and acid fuchsin; but certain others
are occasionally employed. Picric acid forms a few useful com-
pound dyes, rosanilin picrate (i.e., the compound 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.

Ehrlich has 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.

90

CHAPTER IX

THE NATURAL DYES

AS STATED above (p. 11) the group of natural dyes is shrink-
ing as more and more of them are being produced by artificial
means. Alizarin, for example, used to be a natural dye of
much importance; but now the artificial manufacture of this dye
is 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, be-
cause in its chemistry it does not fall in well with any of the groups
of artificial dyes. Indigo is still obtained from the indigo plant,
altho under present-day conditions its artificial manufacture is
ordinarilv the more economical.

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 the difficulty in learning their
exact chemical structure.

The most important natural dyes for the biologist are haema-
toxylin, indigo, cochineal (and its derivatives), orcein, and litmus.

The Indigo Group

INDIGO c. I. NO. 1 17 7

Synonym: Indigo blue.

The plants from which indigo was formerly exclusively manu-
factured are largely species of the genus known as Indigofera, altho
some indigo-bearing plants are recognized by botanists as belong-
ing 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:

CO CO

\ / •

c=c

/ \

NH NH

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.

91

INDIGO-CARMIN C. I. NO. I180

Synonym: Indigotine la.
This is the sodium salt of indigodisulfonic acid :

NaSOj CO CO SOjNa

^rr \-/ ^rr

\/\ / \ /\/

NH NH

Indigo carmin is a blue dye of acid properties, which is sometimes
used as a plasma stain in contrast to carmin, either mixed with it
or following it.*

Cochineal Products c. i. no. 1239

Cochineal is a dye that has long been well known. It is obtained
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.
By treating with alum this solution yields a product somewhat
more free from extraneous matter, known as carmin. This is the
form in which the dye is generalh^ 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 tho less used today than carmin. Alone it has
little value, to be sure, for it has no direct affinity for tissues unless
they contain iron, aluminium or some other metal. It is most
commonly employed either with or following one of these mordants.
A tincture of cochineal, that is an alcoholic solution containing
calcium and aluminium chlorides, has been used by Mayer 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, Czokor, and Partsch;
it can be used for sections, and is specially recommended for stain-
ing 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 for staining sec-
tions. Spuler recommends an iron-alum-cochineal for staining in
bulk when the sections are to be photographed, the technic bring-
ing out nuclei, the blood in the tissues, and the muscle striations;
sections may also be stained by the same method. By this technic
the iron alum is applied first to the tissues as a mordant, and then
followed by the stain. In Hansen's ferri-cochineal, on the other

*For literature references to the procedures listed in this chapter see pp. 110 to
128 and 138 to 1-io.

92

hand, the iron alum is mixed with the dye, and the mixture used for
staining sections of tissue.

Carmin is of considerable historic interest. It was used as early
as 1839 by Ehrenberg, altho as we have seen (p. 7) 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 enormous variety of synthetic dyes now available.
On account of its freedom from toxicity it is useful for staining by
injection. It is much used for staining in bulk, particularly in
embryological work. A well known formula is Schneider's aceto-
carmin, which is a valuable chromatin stain for fresh material in
smear preparations. Alum carmin was used by Grenacher for
similar purposes. Carmin is only slightly soluble in water at a
neutral reaction; so solutions must be either acid (like the two
above) or alkaline. Three alkaline formulae are of considerable
use: ammonia carmin, which has been used both for injection and
for staining sections; soda carmin, used primarily for injection; and
Mayer's magnesia carmin, useful either for sections or for staining
in bulk. Alcoholic solutions are also used: Grenacher's borax car-
min (or as modified by Mayer) being a splendid nuclear stain for
sections; and the hydrochloric carmin of Mayer serving both for
sections and for staining in bulk. A special formula containing
aluminium chloride (known as muci-carmin) has been proposed by
Mayer and is used for staining mucin. In double staining it is
sometimes used with indigo carmin; but most often with picric
acid. Picro-carmin is a very well known combination used for
double staining effects in sections, particularly for nervous tissue;
it stains nuclei red and cytoplasm yellow.

One of the most recent and important uses of carmin is in Best's
carmin 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 haematoxylin. The stain
is permanent; the method is of much importance both to the path-
ologist and to the histologist.

Carminic acid. The dye principle of carmin and cochineal is
carminic acid. This product is obtained by extracting the insect
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

I CO I CeHiiOs

HO / CO f OH
COOH OH

93

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, and has also been used by Grenacher
and Rawitz; it is a useful nuclear stain for sections, and is often
employed with light green or indigo carmin as a contrast stain. A
so-called muci-carmin, an acid solution containing aluminium
chloride, has beem employed by Rawitz to stain mucin; while
Mayer's para-carmine, containing aluminium and calcium chlo-
rides, 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. Carmin, kept in ammoniacal solution, changes in its
properties, due to oxidation. The oxidized carmin, often known as
carmein, can be obtained by treating a carmin solution with hy-
drogen peroxide and precipitating with alcohol. It is a dark colored
mass which can be ground into a black powder.

Orcein and Litmus c. i. no. 1242

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 acids, one of which is
orcin :

OH

/

\
OH

Orcin, acted upon by air and ammonia, becomes orcein.

ORCEIN

The exact formula of orcein is unknown. It is a weak acid,
soluble in alkalies, with a violet color.

In alcoholic solution Unna has used orcein for staining elastin
tissue; he has employed it for connective tissue, following poly-
chrome methylene blue; and for plasma fibrils in the epithelium,
following anilin blue; also with anilin blue or acid fuchsin in study-
ing the process known by him as chromolysis. It has found less
frequent use among other histologists; but has been employed by

94

Israel in acetic acid solution for staining sections (nuclei staining
blue, cytoplasm red); and by Moll, dissolved in weak hydro-
chloric acid, for staining sections of embryos.

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 colored
principle is known as azolitmin.

Litmus scaj-cely needs comment here. It is a feeble dye and is
never used as an histological stain. Its classic use is for indicator
purposes; but it is now coming to be largely replaced by the various
synthetic dyes (especially sulphonphthaleins) which change color
thru an hydrogen-ion range near the neutral point.

Brazilin and Haematoxylin

The two natural dyes, haematoxylin and brazilin are closely
related chemically and upon decomposition yield the two com-
pounds, pyrocatechin

OH

and pyrogallic acid

OH

OH
OH

OH

Both dves are obtained bv extraction of the bark of certain trees,
haematoxylin 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. Haematoxylin
comes from a single species; while brazil wood is a term applied to
various different species all yielding brazilin.

BRAZILIN C. I. NO. I243

The composition of this substance is supposed to be:

HO /\ O CH,

_ C— OH

CH \
I CH.

/ \

\ /

I I
HO OH

95

Its solution is colorless, but it becomes red on exposure to tlje air,
as it is then oxidized into the dye brazilein, which probably has the
formula :

HO

HO O

With alum it is used as a nuclear stain (known as brazalum) by
Mayer. It is also used by Hickson for similar purposes following
treatment with iron alum as a mordant.

HAEMATOXYLIN

C. I. NO. 1246

Haematoxylin is a homolog of brazilin, having one more hy-
droxyl group, the generally accepted formula being:

OH

HO

O CH

CH

C— OH
CH.

<z>

HO HO

Like brazilin it is not a dye, but its color develops in solution upon
standing, due to the oxidation into haematein, which is homologous
to brazilein and probably has the formula:

OH
HO_/\_0_CH.

C— OH
CH.

V

HO O

Haematoxylin 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

96

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 because 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.

Haematoxvlin is seldom used alone, as it has little affinitv for the
tissues in itself, even after "ripening" when it is largely converted
into haematein. Some form of mordanting is ordinarily required;
and most of the haematoxylin 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 haematoxylin alone.
Its greater affinity for plant than for animal tissue implies the
presence of aluminium, copper, or iron in the former. In fact
haematoxylin can be used as a very delicate reagent for iron or
copper.

Perhaps the best known formulae for staining with haematoxy-
lin are the combinations with aluminium, generally in the form of
alum. Bohmer's alum haematoxylin (1865), altho no longer used,
is of much historic interest as it was the first stain of this type to be
used. The best known at present is Delafield's alum haematoxy-
lin, which is very useful tissue stain with great affinity for chroma-
tin and nuclei, and has much value in staining cellulose walls in
vascular plants. Another alum haematoxylin used for similar
purposes is that of Ehrlich.

Mayer's haemalum is another well known alum combination.
In this stain haematein is first prepared and then combined with
alum. The name haemalum, proposed by Mayer, is now generally
accepted for this combination, and various other haemalum form-
ulae have since been proposed. They are useful chromatin stains
and are called for in various special procedures.

Mayer has also combined haematein with aluminium chloride,
his haemacalcium calling for this salt and calcium chloride, while
his muc-haematin contains aluminium chloride and glycerin. The
latter is used for staining mucin.

The iron combinations are perhaps equally valuable. The
original iron haematoxylin was that of Benda; but the best known
at present is M. Heidenhain's, which is one of the most useful
histological and cytological stains, both in botany and zoology. It
is a powerful stain for chromosomes and centrosomes, and is of use
for bringing out the middle lamellae in wood. Various other
modifications of iron haematoxvlin have been used, but thev are
all similar in principle. Ordinarily the iron salt is not mixed with
the stain, but is used for a preliminary mordanting of the tissue.

Haematoxylin has been combined with chromium, one of the
early staining methods being that of R. Heidenhain, which called
for potassium bichromate as a mordant. Various recent modifi-
cations are in use today, such as that of Apathy, for staining general

97

tissue. Weigert uses a chrom combination for staining nervous
tissue.

Benda uses haematoxylin following treatment with a copper salt
for studying spermatogenesis; and Bensley a similar technic for
chromosomes and mitochondria.

Mallory has proposed a formula for haematoxylin containing
phosphomolybdic acid and also one containing phosphotungstic
acid. The latter method is especially valuable for staining 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. Haematoxylin 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 haematoxylin followed by picric acid and
acid fuchsin. A few other methods call for picric acid or ammonium
picrate after haematoxylin; and it is sometimes used with eosin or
after orange G or acid fuchsin. Most of these combinations, how-
ever, are called for only in the case of special procedures.

At present it is possible to obtain very satisfactory haematoxy-
lin manufactured in America. A very good statement of the situa-
tion is given by McClung (1923) . The first American haematoxylin
put on the market, altho pronounced very good by many of those
using it, still proved to be lacking in some of the qualities which the
best haematoxylin ought to possess. Thru the cooperation of the
manufacturers, the trouble was located. In order to obtain a pure
product, SO2 had been used in the manifacture for bleaching pur-
poses; but this treatment was found to be harmful to the haema-
toxylin. The product now on the market is not so treated, how-
ever, but an extra recrystalization is introduced in the place of the
bleaching process; and the resulting product meets all the tests to
which it is submitted.

98

CHAPTER X

THE THEORY OF STAINING

THRUOL T the preceding pages of this book an effort has been
made so far as possible to avoid theoretical discussions. Altho
thev contain some statements the truth of which cannot be
regarded as fully established, as in the case of the chemical com-
position of some of the dyes, the discussion in general has been con-
fined to observations and to chemical information for which there
is good authority, without any attempt to introduce explanations
of a theoretical nature. The present chapter, therefore, was not
part of the original plan of the book, and the decision has finally
been made to introduce it merelv because it is felt that a brief
statement of some of the most probable theories to explain staining
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.

Theories to account for dyeing or staining have in general been
based exclusively upon either physical or chemical phenomena. It
would seem at first thought that the dves combine so firmlv 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, ^^hen 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 possess-
ed; it is, moreover, ordinarily possible to extract all or nearly all of
the color by sufficiently long immersion in water, or by the fairly
brief action of alcohol. Another observation which points against

99

chemical action is that the tissue never removes the dye completely
from solution, even tho very dilute; whereas ordinary chemical
reactions tend to continue until one of the components of the re-
action is exhausted. Such facts as these, to the exponents of the
physical theory, are enough to refute the possibility of chemical
action.

It has, indeed, been pointed out that all of the ordinary dyeing
or staining phenomena can be explained on a physical basis. It is
evident, to be sure, that the action of the dyes is not confined to the
surface of the material colored; but as the substances stained are
always more or less porous, absorption of the dye after passing thru
cell membranes by osmotic action accounts for the penetration. To
account for the selective action of different dyes upon different
parts of the cell it is possible to use the principle of absorption as an
explanation. Adsorption is the property possessed by a solid body
of attracting to itself by purely physical means from a surrounding
solution certain compounds or ions present in that solution. Hav-
ing once entered the tissue it is possible that the dyes remain there
in a state of solid solution, similar to that in which gold is retained
in ruby glass. This possibility, in the opinion of those who hold the
physical theory, is the more likely because 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 solid 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.

Some of those who hold in general to the physical theory of stain-
ing admit that these simple physical phenomena alone cannot ex-
plain everything, as for example, instances in which a dye pene-
trates different cell elements equally readily, but can be easily ex-
tracted from some of them while scarcely at all from others. It is
assumed, therefore, that the dyes penetrate the cells by mere ab-
sorption and diffusion, but are in some cases precipitated there by
acids or bases, or other chemical reagents present, thus preventing
their extraction by simple solvents. Such a theory admits the
possibility of chemical action without assuming an actual chemical
union between the dye and the tissue.

In this connection the action of mordants is interesting. Some
tissues do not stain directly with certain dyes, or if they do the
color is very feeble. If, however, they are treated previously or
simultaneously with certain chemicals, the dyes "take."

It is possible that this phenomenon may be due to chemical
affinities of the mordant for the tissue on the one hand and the dye
on the other; but the special value of iron and aluminium salts as
mordants makes it seem quite probable that their action may be
actually to precipitate the dye in the tissue. To the bacteriologist
the behavior of different bacteria to the Gram stain immediately
suggests itself. In this technic (see p. 68) one of the methyl violet
dyes is allowed to act on the bacteria for a definite length of time,

100

and is then followed by treatment with iodine. After that, alcohol
is applied for decolorization; but it proves that certain kinds of
bacteria retain the violet stain even after counterstaining with some
dye of a different color; while others are readily decolorized by the
alcohol, and take the counterstain. According to some, this action
is accounted for by assuming that the iodine combines with the
methyl violet inside the cell, converting it into a molecule so large
as to be unable to pass thru the cell membrane again. It may, on
the other hand, be assumed that there is an actual chemical differ-
ence between Gram-positive and Gram-negative bacteria, so that
the former combine chemically with the iodine and dye, while the
latter do not. Neither theory has been definitely proved or dis-
proved.

Evidence is still lacking, in fact, to prove or disprove either the
chemical or the physical theory as it relates to general staining. The
difference, perhaps, is not one of immense importance. It is fre-
quently pointed out that there is no sharp distinction between
chemistry and physics, and in such delicate reactions as those in-
volved in staining, we may be well in the borderland between the
two branches of science, where it is impossible 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 phe-
nomenon of staining; and it is these that are considered most im-
portant by the exponents of the chemical theory.

It is pointed out on behalf of the chemical theory that just be-
cause 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 kno^m that some parts of
the cell are acid in reaction, others alkaline; and it is a well kno^m
chemical principle that the former would tend to combine with the
kations in solutions with which they come in contact, the latter
with the anions. Now inasmuch as in certain dyes the color exists
in the kation (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. Argu-
ments for the physical theory which exclude chemical action must
furnish strong proof that no chemical union occurs; and those who
favor the chemical theory claim that such proof is lacking. 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 proved
by the fact that sufficiently long action of solvents removes the
color. Alcohol and even water are not absolutely inert chemically
and may withdraw the dye by chemical instead of physical action ;
the very length of time necessary to remove the color completely
(sometimes so long as to allow bacterial decomposition of the tissue)
indicates that a rather strong union between dye and tissue has

101

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 favor the chemical theory point
out instances where chemical reactions are known to take place
and yet to stop before either component is exhausted; and they
further claim that chemical action is strongly indicated by the fact
that in dilute solutions the tissues take up relatively larger quan-
tities of the dye than in concentrated solutions.

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; tnerefore, 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, as when we find
a certain stain acting only on tde nuclei or even exclusively upon
certain structures within the nuclei. The chemical theory is not
yet firmly established, and the probabilities are that staining is
both a chemical and physical phenomenon; but it is coming to be
more widely accepted than the physical theory. Hence it deserves
a more detailed discussion.

The chemical theory of staining is dependent 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.

Briefly stated, the fundamental chemical theory of staining 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 by this
theory 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
dves and is assumed to be basic in character.

Now this is by no means the whole of the chemical theory of
staining. The theory is so complex and has so many ramifications
and special applications that it takes an intensive study of the
subject to comprehend it fully. It is well knowni for instance, that
certain basic dyes have stronger affinities for certain parts of the
nuclei than for others, and that of the various cytoplasmic struc-
tures outside of the nucleus some are more readily stained by
certain acid dyes and some by others. Such special applications
as these, of course, are not explained on the theory of acid or basic
character alone. It is possible, for example, to use the Flemming
triple stain, which employs the acid dye orange G and the two basic

102

dyes safranin and gentian violet, thus staining the chromatin with
gentian violet and the rest of the nucleus with safranin. It is
difficult to say just how any chemical theory of staining can yet
satisfactorily explain such selective action as this. It is, indeed,
admitted by some upholders of the chemical theory that the chem-
ical action of dyes is not specific, and merely serves to differentiate
acid from basic elements of the tissue; and that the further differ-
entiation, as between chromatin and other parts of the nucleus, is
due to physical forces, thus indicating a difference in the structure
rather than in the chemistry of the different portions of the nucleus.
The neutral stains have a very interesting action. When tissue
is stained with them they seem to break up partlj^ into their com-
ponent acid and basic elements and stain portions of the tissue as
the simple dyes would individually. But in addition the neutral
stain itself seems to have an affinity for certain parts of the tissue
and hence a third color is possible. This explains in part the poly-
chrome effects obtained by the eosin-methylene-blue blood stains —
only partly so, however, because in preparing these stains methy-
lene blue breaks up into certain other dyes so that more shades than
those expected from eosin and methylene blue alone are obtained.
So far as the action of stains is chemical, their use forms a con-
necting link between the two subjects, histology and micro-
chemistry. These two branches of science are generally thought
to be entirely distinct. The histologist, with the technic and view-
point of the biologist, prepares delicate sections of various materials
colored with one or more of a long series of available dyes, and
studies the biological structures present under the microscope. The
microchemist, with the technic and vie^\'point of the chemist,
examines with the microscope similar material treated with various
reagents of known chemical reaction, and from his observations
draws conclusions as to the chemical nature of the substances
examined. There is some possibility, however, that the difference
between histology and microchemistry is one of point of view rather
than of methods. Both the microchemist and the histologist
study the action of chemical compounds on substances or struc-
tures visible under the microscope; the difference is that the micro-
chemist uses the chemical compounds in question as chemical re-
agents while the histologist uses his as dyes to color the microscopic
structures and thus to increase their visibilitv.

Now, on the chemical theory of staining, the biologist is using
complicated chemical reactions in his microscopic technic — so
complicated in fact as to be unintelligible chemically. To bridge
the gap between histology and microchemistry these reactions
must be made intelligible. The first step in this direction has now
been taken by Unna (1921) in a very important contribution to
the subject of cell chemistry. He points out the need of harmoniz-
ing chemical and histological investigations and proposes a method
of doing this which he calls chromolysis. The technic he has de-

103

veloped, altho quite complicated in its details, can be summed up
briefly as follows : To select a dye, or a mixture of dyes, either acid
or basic, which bring out some intracellular structure whose chem-
istry it is desired to learn; then to submit the sections to the action
of various solvents, beginning with simple cold water, next pro-
ceeding to hot water, and from that to the more powerful solvents,
but using only those whose action on proteins, lipoids or carbo-
hydrates is known to the chemist; then to stain the sections with
the staining fluid selected; and finally to determine by microscopic
examination which solvents have removed the substance under
investigation.

By such methods as these Unna hopes to make considerable
progress in the microchemistry of the cell; and it will be readily
seen that once the gap between chemistry and histology is bridged,
progress will become constantly more and more rapid. As soon as
it is possible to obtain reasonable hj'^potheses as to the chemical
nature of the various intracellular bodies in any one particular
type of tissue, then it will be possible to ascertain the affinities of
the different dyes now used in histology for the different chemical
compounds thus recognized; and then by using the same stains on
other tissue it will be possible to apply the information thus ob-
tained to the solution of the chemistry of other microscopic struc-
tures. In other words, stains will become chemical reagents in-
stead of merely dyes for making microscopic structures visible. In
this way it is hoped that chemistry and histology, working to-
gether, may solve some of the obscure problems as to the nature of
the cell and its contents.

.». -'•*'■"

.1'/

i

104

APPENDIX I
TABLES RELATING TO STAINS

In the following tables all the dyes that are frequently mentioned in the literature
dealing with microscopic technic are listed, together with the most important uses
of each in the biological laboratory. The list of uses (Table 2) is necessarily
incomplete. In the case of the most commonly used stains, in particular, it
has been necessary to group various uses together under some general term,
without attempt to list the individual procedures. In general, however, the policy
has been to list the methods for which a stain is most commonly used today, and
of the obsolete methods to give only those of historical interest. Criticism will
be welcomed from anyone noticing any serious omission.

The dyes in Tables 1 and 2 are listed in the same order as in the main part of
this book. Hence either the general Table of Contents or the list in Table 1 may
be used to learn the order of the stains when it is desired to find some particular
one in Table 2. For an alphabetical list referring to the following pages see the
general Index,

The references given in the last column of Table 2 refer ordinarily by name and
date to the literature listed in Appendix III. Five references, however, are used
so often that the date is omitted: they are referred to merely as Chamberlain, Lee,
Ehrl. I, Ehrl. II, and Mai. & Wr. These refer respectively to: Chamberlain's
Methods in Plant Histology (1924), Lee's Microtomists Vade-mecum (1921),
Ehrlich's Enzykopadie der Mikroskopischen Technic (1910), Vol. I and Vol. II,
and Mallory and Wright's Pathological Technic (1924). Xo effort has been made
to give the original reference in all cases ; but rather to refer to some readily available
description of each technic than can be followed by anyone using the procedure.

105

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«3

g

15

o

CO

O
c

CO -be

§ 2<

a

_o o

a

&,
o

be

.s

I

o

&. o
^ :;:
®^

-c 2

't: CO

CX be

e

C

be
.S

!s

CO

Ji

o
Q

Haemetoxylin {continued)

128

Table 3. A List of Biological Stains Grouped According to the Field in

Which Used.*

animal histology
Nuclear stains (basic)

Janus green B.

Thionin

Methylene blue

Toluidin blue

Cresyl violet

Safranin

Magdala red

Auramin

Fuchsin

Hoffman's violet

Iodine green

Gentian violet (including'crystai and methyl violets)

Cochineal and carmin

Orcein

Brazilin

Haematoxylm

Cytoplasm stains (acid)

Picric acid
Orange G.

Bordeaux red

Fast yellow (bone tissue)

Methyl orange (for keratin in skin)

Amaranth (nervous tissue)

Biebrich scarlet W. S.

Bismarck brown

Congo red

Tr^'pan red (vital staining)

Benzopurpin -iB

Tr\T)an blue (vital staining)

Alizarin red S (for nervous tissue)

Neutral red (largely for vital staining)

Nigrosin

Malachite green

Light green SF yellowish

Acid fuchsin

Methyl blue

Anilin blue W. S.

Rhodamine

Eosin Y.

Eosin, ale. sol.

Erythrosin (nervous tissue)

Indigo carmin

Fat stains

Sudan III
Sudan IV

Nile blue sulfate.

*In this table the stains in bold faced type are those of widest application.
When a stain is specified for practically only one purpose that purpose is mentioned
in parenthesis; the stains not so designated are of fairly general application in the
particular field under which they are listed.

U9

PLANT HISTOLOGY

For lignified cell walls

Methylene green

Safranin

Iodine green

Gentian violet (including crystal and methyl violets)

Methyl green

For cellulose walls

Congo red (plant mucin)

Malachite green (for host tissue in case of fungus diseases)

Light green SF yellowish (cellulose walls)

Acid fuchsin

Eosin Y

Erythrosin.

Haematoxylin

CYTOLOGY

General nuclear stains (basic)

Thionin

Methylene blue

Toluidin blue

Magdala red

Fuchsin

Gentian violet (including crystal and methyl violets)

Methyl green

Carmin

Orcein

Haematoxvlin

Special chromatin stains

Alizarin red S

Thionin

Methyl green

Safranin

Gentian violet (including crystal and methyl violets)

Iodine green

Carmin (foi fresh cells)

Haematoxvlin

Cytoplasm stains (acid)

Picric acid
Orange G.

Bordeaux red
Methyl orange
Acid fuchsin
Eosin Y.

Stains for mitochondria, etc.

Aurantia
Janus green B
Acid fuchsin
Crystal violet.

130

PATHOLOGY AND BACTERIOLOGY

Nuclear stains (basic)

Thionin
Methylene blue

Toluidin blue

Safranin

Cresyl violet

Fuchsin

Hoffman's violet

Iodine green

Gentian violet (including crystal

and methyl violets)
Pyronin

Cochineal and carmin
Orcein
Haematoxylin

Blood stain constituents

Orange G. (acid)
Narcein (acid)
Methylene blue (basic)
Methylene azure (basic)
Neutral red (acid)
Acid fuchsin (acid)
Methyl green (basic)
Pyronin (basic)
Rhodamine (acid)
Eosin Y (acid)

Fat stains

Sudan IV

Nile blue sulfate.

Bacterial stains

Cytoplasm stains (acid)

Picric acid

Martins yellow (for cancer

tissue)
Orange G.
Methyl orange

Amaranth (for nervous tissue)
Biebrich scarlet W. S.
Congo red
Alizarin red S.
Neutral red
Nigrosin

Malachite green Used

Light green SF yellowish
Acid fuchsin
Methyl blue
Anilin blue, W. S.
Eosin Y.
Ervthrosin

Bismarck brown (Gram counter-
stain)

Thionin

Methylene blue

Safranin (Gram counterstain)

Fuchsin

Gentian violet (including crystal
and methyl violets)

Methyl green (constituent of
Pappenheim stain)

Pyronin

Erythrosin

Rose bengal

in bacteriological media

Neutral red
Fuchsin
Acid fuchsin
Brilliant green

Methylene blue
Eosin Y.

131

APPENDIX II
COMMISSION SPECIFICATIONS OF CERTAIN STAINS

As announced in several notes published by the Commission in Science, it is
planned to draw up specifications for all the stains put on a certification basis.
So far as these specifications have been prepared they are given on the following
pages. Similar specifications for other stains will be prepared later.

It must be understood that these specifications are not intended to furnish
definite statements as to the chemistry of satisfactory stains. The object with
which they were drawn up was to allow the manufacturers as much latitude in the
matter of chemical composition as has been found consistent with good results in
practice, and to lay the greatest stress upon the performance of the stains in actual
laboratory use. The requirements listed are those which must be met by dyes
submitted to the Commission for certification.

These specifications are published, moreover, with the understanding that they
are subject to revision at any time. Further investigations are in progress con-
cerning the adaptability of various dye products for different purposes, and also
as to the relation of optical characters to performance in staining. The Commission
reserves the right to modify the specifications for any stain in regard to either of
these two points as data accumulate to show the need of modification.

SPECIFICATIOXS FOR METHYLENE BLUE

1. Samples of methylene blue to be considered must be of the so-called medicinal
grade. It is expected that they will meet the U. S. P. requirements, but less weight
will be attached to this consideration than to those following. In other words, a
sample giving satisfactory performance will not be excluded because of failure in
some particular to meet these chemical requirements.

2. Methylene blue for the purpose above specified must contain at least 75
per cent total color, this to be determined by reduction with titanous chloride.
When reduced by titanous chloride in an atmosphere of carbon dioxide, 1 gram of
the dye must consume at least 4.69 cc. normal titanous chloride solution.

3. The methylene blue must have no solvent action on casein. This is to be
determined as follows: Prepare two 1 per cent solutions of this stain, one in distilled
water, the other in tap water. Place single drops of skimmed milk on each of two
clean glass slides and smear each drop over a surface of about one square centimeter
so as to form a very thin film of milk; allow this film to dry without heat or at a
temperature not over 60°C., immerse for about a minute in xyol to dissolve the fat,
then for the same length of time in alcohol to coagulate the casein. After this
immerse one slide in the distilled water solution of methylene blue and the other
slide in the tap water solution, allowing them to stand for three minutes; at the
end of this period there should be no action of the stain on the casein.

4. The methylene blue should stain the diphtheria organism in any of the types
of solution^ ordinarily employed. It should be tested as follows: Prepare three
solutions of the stain, one a 1 per cent solution in distilled water, the second a
mixture of three parts saturated alcoholic solution to 10 parts of distilled water,

1S2

and the third three parts of saturated alcoholic solution to 10 parts of 0.01 per cent
NaOH. Prepare three slides of a fresh culture of a diphtheria organism; stain one
slide in each of these three solutions for two or three seconds only, i. e., just as
briefly as the stain can be poured on and poured off, and wash each slide im-
raediatly. Examined under the microscope all three of these preparations should
show deeply stained bacteria with the characteristic metachromatic granules
suflSciently distinct to insure accurate diagnosis.

5. The sample should prove satisfactory for histological use. Xo exact method
for determining this can be given, but the sample must be submitted to one or two
experts in histological technic in order to get their judgment.

6. It must be understood that these standards refer to samples to be used for
ordinary bacteriological and histological staining. Special standards for methylene
blue used in vital staining will undoubtedly be necessary. These standards, how-
ever, have not yet been determined.

SPECIFICATIONS FOR SAFRANIN' O

1. Samples of safranin O must be of the t^-pe represented by Colour Index
No. 841 and on spectrometric analysis .should have an absorption curve maximum
at approximately olou/x as determined in a one cm. layer by a spectrophotometer.
Other dyes must not be present.

2. Safranin samples to be certified by the Commission must contain at least 75
f>er cent total color as determined when reduced V>y titanous chloride in an atmos-
phere of carbon dioxide. One gram of the dye must consume at least 4.195 cc.
normal titanous chloride solution.

3. The samples should prove satisfactory for histological use. No exact method
for determining this can be given, but the samples must be submitted to one or two
experts in histological technic in order to get their judgment. Their judgment
must be based to a considerable extent upon the behavior of the stain in the Flem-
ming triple staining technic, in which it is used together with orange G and gentian
violet. In other words, the stain must be of such a shade as to contrast well with
both of these two other dyes.

SPECIFICATIONS FOR BASIC FUCHSIN

1. Basic fuchsin designed for staining and indicator purpo.ses must be ro.saniliu
or new fuchsin (Colour Index No. 678) or else a mixture of rosanilin and para-
rosanilin containing at least half of the former (that is, c-orresponding to Colour
Index No. 677).

2. Fuchsin samples to be certified by the Commission must be of such a strength
that, when reduced by titanous chloride in an atmosphere of car])on dioxide, one
gram of the dye will consume at least 46.5 cc. normal titanous chloride solution.
A sample of this strength will be between 76 and 85 per cent total dye content, the
exact dye content varying according to the relative amounts of the higher and the
lower homologs present.

8. The sample should prove satisfactory for staining the tuhercle organism and
should retain its color sufficiently when treated by the Ziehl metliod t» be diagnostic
when staining tubercular discharges. This must be determined by an investigator
skilled in this particular technic.

133

4. The sample must prove satisfactory for use in the Endo medium. In making
this test the following technic should be used: A saturated alcoholic solution is
diluted 2 to 3 times, the dilution to be such that no precipitation occurs when mixed
with a sodium sulphite solution. Then add 0.5 cc. of the saturated and the diluted
fuchsin solutions each to 10 cc. of a 2.5 per cent sodium sulphite solution. Select
the strongest of these which shows no precipitate and add it to the other ingredients
of Endo agar, sterilize and cool. It should then be colorless, but the color must be
restored by the colon and dysentery organisms when inoculated upon it. The test
must be made by one familiar with the technic in question.

5. It must be understood that as basic fuchsin is used in other special forms of
technic, new standards may be called for. The present specifications apply par-
ticularly to the above mentioned two uses: but samples fulfilling them are ordi-
narily satisfactory for all histological purposes.

SPECIFICATIONS FOR ACID FUCHSIN

1. Acid fuchsin designed for staining and indicator purposes must correspond
to Colour Index No. 692. Inasmuch as the dyes of this type on the market are
mixtures of greatly varying composition, and it has not yet been shown that any
one of the components of these mixtures is better for biological purposes than any
of the others, no definite physico-chemical specifications are made at the present
time, with the reservation that more specific requirements may be drawn up at a
later date if future work shows them to be desirable.

2. Acid fuchsin samples to be certified by the Commission should be of such a
strength as to consume not less than 2.0 cubic centimeters of normal TiCls solution
per gram of sample. Such samples would have a total dye content of approximately
60 per cent.

3. Acid fuchsin samples for certification should prove satisfactory counter-
stains in general histological work. They should in particular prove satisfactory
in the Bensley technic for staining mitochondria, and in the Mallory connective
tissue stain (with anilin blue and orange G). They should be tested for these
purposes by histologists skilled in the technic in question.

4. The samples shall prove satisfactory for use in the Andrade indicator. For
this purpose they shall be tested as follows: Dissolve 0.2 gram in 100 cc. water,
neutralize by adding between 15 and 25 cc. of Normal NaOH. When neutralized
with the proper amount of the reagent, the solution should become a straw color
upon standing and should impart no noticeable color to ordinary bacteriological
nutrient agar when added at the rate of 1 per cent. The red color should be
restored in this medium by the colon organism when growing in the presence of
any sugar attacked by it.

5. Labels to be used on the acid fuchsin samples should state the formula giving
best results with the sample in question in preparing the Andrade indicator.

SPECIFICATIONS FOR GENTIAN AND CRYSTAL \^OLET

1. Crystal violet samples shall contain no dye except hexa-meth\l pararosanilin
(Colour Index No. 681), having its absorption maximum at 590fifi when de-
termined in a cell of 1 milimeter thickness in a spectrophotometer.

134

i. Crystal violet samples to be certified by the Commission must be of at least
80 per t!ent dye content as determined by reduction of TiCls in an atmosphere of
carbon dioxide.

3. Gentian violet for general staining purposes, as defined by the Commission
on the Standardization of Biological Stains, must be either penta-methyl or hexa-
methyl pararosanilin, or else a mixture of methylated pararosanilins composed
primarily of the two compounds just named and having a shade at least as deep
as that recognized in the trade as methyl violet 2B. In other words the dye must
be Colour Index Xo. 680 or No. 681.

4. Gentian violet samples to be certified by the Commission must be of at least
75 per cent dye content as determined by reduction of TiCl3 in an atmosphere of
carbon dioxide. The diluent used must be dextrin.

5. The sample of crystal or gentian violet should prove satisfactory for staining
bacteria according to the Gram technic when made up in a 0.5 per cent aqueous
solution, without the use of anilin oil or any other mordant, and stained by the
following procedure: crystal or gentian violet 1 minute, Lugol's iodine solution 1
minute, 95 per cent alcohol 30 seconds, 0.05 per cent safranin solution 10 seconds.
In making the test, a weakly Gram-positive orgnism and a Gram-negative organism
should be tested.

6. The sample should prove satisfactory in the Flemming triple stain when
used with safranin and orange G. For this purpose it must be tested by histologists
skilled in the technic in question. It should also prove a satisfactory nuclear
stain in general histology.

7. Either gentian violet or crystal violet for certification must have sufficient
bacterostatic power so that, when added to nutrient agar in the proportion of one
part to a million, it will entirely prevent the growth of Bacillus suhtilis when this
organism is streaked over the surface of the hardened agar. It must be understood,
however, that this bacterostatic property of the dye is still under investigation
and has a relation not yet fully understood to the control of certain diseases. As
new investigations bring new light on the subject it may be necessary to draw up
new specifications to cover this point.

SPECIFICATIONS FOR PYRONIN

1. Pyronin designed for staining purposes must be either pyronin G (Colour
Index No. 739) or pyronin B (Colour Index No. 741). The container in which it is
sold should be marked plainly as to which of these two dyes is furnished.

2. Samples of pyronin G must be characterized by an absorption curve showing
a maximum absorption at about oiSfifx Avhen determined in a 0.002 per cent solution
in a layer 1 milimeter thick. To be certified by the Commission these samples
should be of such a strength that the extinction coefficient at the point of maximum
absorption shall be not less than 1.20.

3. Samples of pyronin B tested in the spectrophotometer under the above
mentioned conditions must have an absorption maximum at about 550;a/x, and must
be of such a strength that the extinction coefficient at the point of maximum ab-
sorption shall be not less than 1.00.

4. The samples shall be satisfactory for use in the Pappenheim combination
with methyl green. In the case poor results are obtained with the formulae for

135

this stain commonly found in the literature it should be suspected that the trouble
might be due to the greater concentration of pyronins now available and the tests
should be made by the following formula calling for a suialler proportion of pyronin :

Methyl green 100 gm.

Pyronin 25 gm.

Alcohol o. cc.

Glycerin 20 cc.

,2 per cent carbolized water 100 cc.

SPECIFICATIONS FOR EOSIN Y

1 . vSamples of eosin Y must be of the type represented by Colour Index No. 768.
They must, furthermore, be the dibasic sodium salt of tetra-brom fluorescein
having an absorption maximum at approximately 516^;* as determined in a .002
per cent solution in a layer 1 cc. thick in a spectrophotometer. They should be
readily soluble in water and alcohol (insoluble material not over 0.5 per cent) .

2. Eosin samples to be certified by the Commission must contain at least 85
per cent total color as determined when reduced by titanous chloride in an atmos-
phere of carbon dioxide.

3. Eosin samples must yield a satisfactory Wright's stain in combination with
methylene blue by the formula given in Mallory and Wright (1924) page 470.
The quality of the resulting compound d\'e must be judged by someone skilled in
its use for staining blood.

4. The samples must prove satisfactory for counterstaining against basic dyes
in histological technic, their performance to be judged by experts in the staining
procedures involved.

5. Eosin should prove satisfactory with methylene blue in the Levine eosin-
methylene-blue medium for the differentiation of certain organisms of the colon
typhoid group. In making this test the method should be followed wliich is
published by Levine (1921) p. ()2-4.

SPECIFICATIONS FOR ORANGE G

1. Samples of orange G submitted for certification must be of the t^^pe listed in
the Colour Index as No. 27 and must be characterized by an absorption maximum
at approximately 485/A/i as determined in a .004 per cent solution in a layer 1 cc.
thick in a spectrophotometer.

2. The total dye content of the samples must be at least 80 per cent as de-
termined by reduction of titanous chloride in an atmosphere of carbon dioxide.

3. The samples must prove satisfactory for counterstaining in histology and
cytology, giving a clear orange shade and in a brown tone to the cytoplasmic bodies.
They shall also be satisfactory for the Flemming triple stain, in which this dye is
used in contrast to safranin and crystal violet. Its performance in these procedures
shall be judged only by ones skilled in the technic in question.

SPECIFICATIONS FOR HAEMATOXYLIN

1. A sample of haematoxylin to be considered for certification must consist of
well defined crystals showing sandy or light brown color and shall contain less than
0.1 per cent ash. With NaOH it shall yield a purple solution turning brown on

1S6

standing in presence of the air; with lead acetate it shall yield first a colorless and
then a blue precipitate darkened by the air.

2. When used by one of the standard methods such as that of Haidenhain for
staining actively growing root tips or other good cytological material showing
mitosis, it shall give a sharp, vigorous black staining of the chromosomes. When
used in the Delafield method, it must give a clear-cut blue picture of chromatic
material. These tests shall be made by someone skilled in the technic involved.

3. Solutions shall show no discoloration and shall retain their staining qualities
upon standing for a period of three or four weeks.

SPECIFICATIONS FOR THIONIN

1. Samples of thionin (Syn. Lauth's violet) must correspond to Colour Index
No. 920 and be characterized by an absorption curve Avith a maximum at about
aOififi as determined in a ,001 per cent solution in a layer 1 cc. thick in a spectro-
photometer.

2. Samples submitted for certification must have a dye content of at least
85 per cent as determined by titanous chloride reduction in an atmosphere of
carbon dioxide.

3. The samples should prove satisfactory for staining frozen sections of fresh
animal tissue and should show good metachromatic effects when applied in a 1
per cent solution for 1 to o minutes, followed by rapid washing and mounting in
water. Their performance in this matter shall be judged by someone familiar with
the technic.

4. The samples should prove satisfactory for staining bacteria by the "little
plate" technic described by W. D. Frost in Jr. Inf. Dis. 19, (1916) p. 273-287

137

APPENDIX III
BIBLIOGRAPHY

(Matter in parenthesis indicates the purpose for which each reference is cited in the
preceding pages, not necessarily the main subject matter of the article in question.)

Anonymous.

1865. Injectionsmassen von Thiersch und MuUer, Arch. Mikr. Anat., 1, llfS.
(Use of carrainates with oxalic acid.)
Albert, H.

1920. Diphtheria bacillus stains with a description of a "new" one. Am. J.
Pub. H., 10, 334- (Toluidin blue and methyl green for staining
diphtheria preparations.)
Ambler, J. A., and Holmes, W. C.

1924. The investigation of biological stains in the Color Laboratory of the
Bureau of Chemistry. Sci., 60, 501-502.
Benda, Carl.

1891 . Neue Mittheilungen uber die Entwickelung der Genitaldriisen and uber
die Metamorphose der Samenzellen. Arch. f. Anat. u. Phys. {Phys.
Aht.) 1891, 5Jf9-552. (Safranin with light green for staining
spermatozoa.)
1899. Weitere Mittheilungen liber die Mitochondria. Arch.f. Anat. w. Phys.
{Phys. Aht.) 1899,376-383. (Proposes crystal-violet-alizarin method
for chondriosomes.)
1901. Die Mitochondriafarbung und andere Methoden zur untersuchung der
Zellsubstanzen. Anat. Anz., Ergdnzhft. 19, 155-17Jf. (Describes the
crystal- violet-alizarin method.)
Beneke.

1862, Correspbl. d. Ver.f. gemeinsch. Arheiten, No. 59, 980. (A note without
title, being first reference to use of anilin dyes in histology.)
Bensley, R. R.

1911. Studies on the pancreas of the guinea pig. Am. J. Anat., 12, 297-388.
(Acid fuchsin and Janus Green for chondriosomes. Describes
"neutral gentian.")
Bergonzixi, C.

1891. tjber das Vorkommen von granulierten basophilen und acidophilen
Zellen im Bindegewebe und uber die Art. sie sichtbar zu machen.
Anat. Anz., 6, 595-600. (Methyl orange in place of Orange G in
Ehrlich-Biondi stain.)
Bernthsen, a.

1885. Studien in der Methvlen blau gruppe. Liebig's Ann. de. Chimie., 230,

73-136, 137-211. ^(Chemistry of Azur I, etc.)
1906. Ueber die chemische Natur des Methylenazurs. Ber. d. Deut. Chem,
GesselL, 39, II, 1804--1809.
Best, F.

1906- Ueber Karminfarbung des Glycogens und der Kerne. Zts. Wis. Mikr.,
23, 319-322.

BOHMER, F.

1865. Zur pathologischen anatomic der Meningitis cerebromedullaris epi-
demica. Aerztl. Intelligenzb. {Munich). 12, 539-548. (First use of
alum haematoxylin.)

BOTTCHER, A.

1869. Ueber Entwickelung und Ban des Gehorlabyrinths nach untersuchungen
an Saugethieren. I Theil. Verh. Kais. Leop.-Carol. deut. Akad.
Naturf., Dresden, 35, Abh. No. 5, pp. 1-203. (First use of alcohol for
differentiation after staining.)

138

Brasil, Louis.

1905. Sur la Reproduction des Gregarines monocystidees. Arch, de Zool.

Exper. et Gen., ^ Ser., 4, 69-100. (Light green with haematoxylin for
sections of seminal vesicles.)
Browning, C. H., Gilmore, M., and Mackle, T. J.

1913. The isolation of tjTjhoid bacilli from feces by means of brilliant green
in fluid media. J. Hyg., 13, 33o-3It2.

Carnoy, J. B., and Lebrux, H.

1897. La fecondation chez I'Ascaris megalocephala. La Cellule, 13, 63-195.

(Congo red with haematoxylin and other nuclear stains.)

Chamberlain, C. J.

192-i. Methods in Plant Histology. Fourth edition, xi and 3^9 pp. Univ. of
Chicago Press.

ClACCIO, C.

1906. Rapporti istogenetici tra il simpatico e le cellule cromaffini. Arckivio.

Ital. di. Anat. e. d. Embriol , 5, 2-56-267. (Iodine green with acid
fuchsin and picric acid for nervous tissue.)

Conn, H. J.

1921. Rose bengal as a general bacterial stain. /. Bad., 6, 253-25^.
CORTI, A.

1851. Recherches sur I'organe de I'ouie des mammiferes. Zts. Wis. Zoo!., 3,
109-169. (An earlv use of carmin in histologv, see note 10, p. 143-4).
Cyon, E.

1868. L'eber die Xerven des Peritoneum. Ber. d. k. Sachs Gessel. d. Wiss. zu
Leipzig., 20, 121-127. (Gives technic of carmin staining.)

CZOKOR, J.

1880. Die Cochenille-Carmin losung. Arch. Mik. Anat., 18, ^12-Ifl4- (Uses
alum cocchineal.)
Daddi, L,

1896. Xouvelle Methode pour colorer la graisse dans les tissues. Archives
Ital. de Biol, 26, H3-146. (Proposes Sudan HI.)
V. DiGRALSKi, W., and Coxradi, H.

1902. Ueber ein Verfahren zum Nachweis der t>T)hus bazillen. Zts. f. Hyg.
39, 283. (Uses crystal violet in agar.)

Ebbinghaus, Heinr.

1902. Eine neue Method zur Farbung von Hornsubstanzen. Ctblt.f. Allgem.
Path. u. Path. Anat., 13, ^22-426. (Methyl orange for keratin.)

Ehrlich, P.

1910. Enzyklopadie der Mikroskopischen Technik. Second Edition. Urban &
Sckwartzenberg, Berlin. Vol. 1, 800, pp.; 2, 680, pp.

Ehrltch, p., and Lazarcs, A.

1898. Die Anaemic. I Abt. In NothnageVs Spec. Path. u. Ther., Bd. 8,

Vienna. (Describes various "neutral" stain mixtures; the "triacid"
mixture; also pyronin and narcein with methyl green or methylene
blue; and narcein with acid fuchsin and methyl green.)

Ehrenberg, C. G.

1838. Die Infusionsthierchen als volkommene Organismen. Leipsig.
Endo, S.

1904. Ueber ein Verhaften zum Nachweiss der Typhusbacillen. Cenibl. f.
Bakt., I Abt., Orig., 35, 109-110. (Proposes the fuchsin agar known
as "Endo medium.")

Faris, H. a.

1924. Neutral red and Janus green as histological stains. Anat. Rec, 27,
2U-2U-
Fischel, Alfred.

1901. Untersuchungen liber Vitale Farbung. Anat. Hefte. I Abt. Bd. 16,
No. 3ik {Hjte. 52/3) U7-519. (Auramin for salamander larvae.)

139

Flemming, W.

1881. Ueber das E. Herraann'sche Kernfarbungs verfahren. Arch. Mikr.

Anat., 19, 317-330. (Magdala red as a nuclear stain. Investigated
principle of differentiation with alcohol.)

1884. Mittheilungen zur Farbetechnik. Zts, Wis. Mikr., 1, 349-361.

1891. Ueber Theilung und Kernformen bei Leukocyten, und iiber denen
Attractionsspharen. Arch. Mik. Anat., 37, ^49-298. (Triple staining
technic — gentian violet, safranin and orange G — described on p. 296.)
Foot, Katherine and Strobell, Ella Church.

1905. Prophases and Metaphases of the first maturation spindle of Allolobo-
phora foetida. Am. J. Anat., 4, 199-2If3. (Use of Bismarck brown
for staining chromosomes in smear preparations of eggs.)
Frey.

1868. Die Hamatoxylin farbung. Arch. Mikrosk. Anat., 4, 3^5-6. (First
use of alum and haematoxylin in a single solution.)
Frost, W. D.

1916. Comparison of a rapid method of counting bacteria in milk with the

standard plate method. ./. Inf. Dis., 19, 273-287. (Use of thionin

for staining young bacterial colonies.)
Gerlach, J.

1858. Mikroskopische Studien aus dem Gebiet der menschlichen Mor-
phologic. Erlangen, 1858. pp. 72. (Shows the advantage of dilute

carmine solutions.)
Giemsa, G.

1902. Farbemethoden fiir Malariaparasiten. Centbl. f. Bakt., I Abt., 32,

307-313. (Describes use of Azur I and Azur II.)
1902. Farbemethoden fiir Malaria parasiten. Centbl. f. Bakt., I Abt., 31,

.'f29-J^30. (Showing the value of preparing blood stains with eosin

and Azur I alone.)

1904. Eine Vereinfachung und Vervollkommnung meiner Methylen-azur-

Methelen-blau-eosin Farbemethode zur Erzielung der Romanowsky-
Nochtschen Chromatinfarbung. Centbl. f. Bakt., I Abt., 37, 308-311.
Gierke. H.

1884, 1885. Farberei zu mikioskopischen Zwecken. Zts. Wis. Mikr., 1,
62-100, 372-m, m-557, 2, 13-36, 16^-221. (Discussion of history
of staining.)
Goppert, H. R., and Cohn, F.

1849. Ueber die Rotation des Zellinhaltes von Nitella flexilis. Botan. Zeitg., 7,
665-673, 681-691 , 69 7-705, 713-719. (First use of carmin for micro-
scopic staining purposes.)
Gr.\m, C.

1884. Ueber die isolierte Farbung der Schizomyceten in Schnitt imd
Trochenpraparaten. Fortschritte der Med., 2, 185-189.
Grenacher, H.

1879. Einige Notizen zur Tinctionstechnik, besondes zur Kernfarbung.

Arch. Mikr. Anat., 16, Ji63-J^71. (Uses alum carmin.)
Griesbach, H.

1882. Ein neues Tinctionsmittel fiir menschliche und thierische Gewebe.

Zool. Anz. 5, 406-^10. (Iodine green as a nuclear stain.)

1880. Weitere Untersuchungen iiber Azofarbstoffe behufs Tinction mensch-

licher und thierischer Gewebe. Zts. Wis. Mikr., 3, 358-385.
(Congo red and amaranth for staining axis cylinders.)

GUYER, M. F.

1917. Animal Micrology. Revised Edition. 289 pp. University of Chicago

aTCSS
IL\NSEN, F. C. C.

1905. Ueber Eisenhamatein, Chromalaumhamatein, Tonerdealaunhamatein,

Hamateinlosungen und einige Cochenille farblosungen. Zts. Wis.
Mikr., 22, 45-90.

140

Hartig, Th.

1854. Ueber die Functionen des Zellenkerns. Botan. Zeitg , 12, o7If-58It.

(A comprehensive investigation of the ability of various parts of

plant protoplasm to take carmin.)
1854. Ueber das Verhalten des Zellkerns bei der Zellentheilung. Botan. Zeitg.,

12, 893-902. (An early use of carmin.)
1858. Entwickelungsgeschichte des Pflanzenkeims, dessen stoffbildung

wahrend der Vorgange des Reifens und des Keiraens. Leipsig, 1858,

16Jt pp. (Uses carmin.)

Hbidenhain, R.

1888. Beitrage zur Histologic und Physiologic der Diinndarmschleimhaut.
Pflug. Arch. Ge.s. Physiol, 43, Supple., 103 pp.

Hebmann, E.

1875. Ueber eine neue Tinctionsmethode. Tagbl. d. IfS Versaml. deut. Naturf.
u. Aerzte, Graz 1875, 105 pp. (Early use of alcohol differentiation
to bring out nuclei.)

Hjcksox, S. J.

1901. Staining with Brazilin. Qu. .J. Micr. Sci., N. S. 44, J^69-J^71. iBrazUin
after iron alum.)
HucKER, G. J. and Conts, H. J.

1923. Methods of Gram staining. .V. Y. Agric. Exper. Sta. Tech. Bid. 93.
Jenner, Louis.

1899. A new preparation for rapidly fixing and staining blood. Lancet, 1899,

Pf. 1. 370.
Kehrmaxx, F.

1906. Ueber Methylen-azur. Ber. d. Deut. Chem. Gessell., 39, II, 1W3-1W8.
Klbbs, G.

1886. I.Teber die Organisation der Gallerte bei einige Algen und Flagellaten.
Unter. Bot. Inst. Tubingen. 2, No. 2, 333-^18. (Congo red as reagent
for cellulose: see p. 369.)

KULTSCHITZKY, N.

1895. Zur Frage uber den Ban der Milz. Arch. Mik. Anat., 46, 673-695.
(Magdala red for elastic tissues.)
Lee, A. B.

1921. The Microtomists Vade-mecum. Eighth Edition, edited by .J. B.
Gatenhy. Blahistons, Philadelphia.
Lee, a. B., and Mayer, P.

1907. Grundzuge der mikroskopischen Technik. Third Edition . Berlin. 1907.
Leishmanx, W. B.

1901. A simple and rapid method of producing Romanowsky staining in
malarial and other blood films. Brit. Med. J., 1901, Pt. 2, 757-758.
(Redissolved precipiate of Xocht stain in methyl alcohol.)
Levine, M.

1921. Bacteria fermenting lactose and their significance in water analysis.

Iowa Engineering Exp. Sta. Bui. 62. (Use of eosin-methylen-blue
agar; see p. 62-4.)
List, J. H.

1885. Zur Farbetechnic. Zt^s. Wis. Mikr., 2, lJi^5-156. (Uses eosin preceding
methyl green.)
MacNeal, W. J.

1906. Methylene violet and Methylene azur. ./. Inf. Dis., 3, U2-It33.
(The history of blood stains and the chemistry of its ingredients.)

1922. Tetrachrome blood stain; an economical and satisfactory imitation of

Leishmann's stain. J. Am. Med. Assn., 78, 1122.
1925. Methylene violet and methylene azure A and B. ./. Inf. Dis., 36,
538-5^6.
Maijx)ry, F. B.

1900. A contribution to staining Methods. J. Exp. Med., 5, 15-20. (Anilin

blue connective tissue stain described.)

141 ^

1904. Scarlet fever. Protozoan-like bodies found in four cases. /. Med. Res.,
10, Jf83-Jt92. (Eosin as contrast stain to methylene blue for tissue,
especially in pathology.)
Mallory, F. B., and Wright, J. H.

1924. Pathological Technic. Eighth Edition. Saunders, Philadelphia.
Mann, Gustav

1902. Physiological Histology. Clarendon Press, Oxford.
Maschke, O.

1859. Pigmentlosung als Reagenz bei Mikroskopisch phj^siologisch XJnter-
suchungen. Bot. Zeitg., 17, 21-27. J. f. prakt Chem. v. Erdmannu.
Wether, 76, 37. (First use of indigo.)
Mayer, Paul.

1878. Die Verwendbarkeit der Cochenille in der microscopischen Technic.
Zool. Anz., 1, 3If5~6. (Uses Cocchineal with alura.)

1891. Ueber das Farben mit Hamatoxylin. Mitt a. d. Zool. Stat. Neapel, 10,

170-186. (Haemalura, haemacalcum, etc.)

1892. Ueber das Farben mit Carmin, Cochenille und Hamatein Thonerde.

Mitt. a. d. Zool. Stat. z. Neapel, 10, J^80-504.
1896. Ueber Schleimfarbung. Mitt. a. d. Zool. Stat. z. Neapel, 12, 303-330.
(Describes muci-carmine, muc-haematin and gluchaematin.)

1899. Ueber Hamatoxvlin, Carmin, and verwandte Materien. Ztsch. Wis.

Mik., 16, 196-220.

McClung, C. E.

1923. Haematoxylin. Sci., 58, 51'.

MiCHAELIS, L.

1900. Die vitale Farbung, eine Darstellungsmethode der Zellgranula. Arch.

Mikr. Anat., 55, 558. (Uses Janus green for chondriosomes.)

1901. Ueber Fett Farlistoffe. Virchows Arch. J. Path. Anat. u. Phys., 164.

263. (Proposes Sudan IV) .
MiJLLER, H. A. C.

1912. Kernstudien an Pflanzen. Arch. f. Zelljorschiing. 8, Hft. 1, 1-61.
(Applies to plant pathology the stain mixture of Pianese — malachite
green, acid fuchsin and martins yellow.)

NOCHT.

1898. Zur Farbung der Malariaparasiten. Centbl. f. Bakf. I Abf., 24, 839-843.

(First to polychromize methylene blue intentionally in preparing
blood stains.)

Osborne, S. G. .

1857. Vegetable cell structure and its formation as seen in the early stages of
the growth of the wheat plant. Trails: Micro. Soc, 5, 10^-122.
(Observes coloring of cell contents in plants grown in colored solu-
tions— carmin. indigo, or vermillion.)

Pal.\dino, Giovanni.

1895. Delia nessima partecipazione dell' epitelio della mucosa uterina e della
relative glandole alia formazione della decidua vera e riflessa nella
donna. Rend. d. Accad. Sci. fisiche e matemat. Napoli, 34, p. 208-S15.
(Biebrich scarlet with alum haematoxylin.)

Pappenheim, a.

1899. Vergleichende Untersuchungeniiber die elementare Z usammensetzung

des rothen Knochenmarkes einige Saugethiere. Virchow\s Arch. /.
Path. Anat. v. Phys.. 157, 19-76. (Uses mixture of methyl green
and pyronin.)

Pelagetti, M.

1904. Ueber einige neue Farbungsmethoden mit Anwendung der Zenkerschen
Fi.xierungsfliissigkeit in der histologischen Technik der Haut-
Monatsch. f. Prak. Dcrmat., 38, p. 532-536. (Biebrich scarlet after
polcyhrome methylene blue or after Unna's haematein. Rose bengal
following haematoxylin.)

142

Peter, Karl.

1899. Die Bedeutung der Nahrzelle im Hoden. Arch. Mirk. Anai., 53, 180-
211. (Light green with haematoxj'lin.)

PfITzer, E.

1883. Ueber ein Hartung und Farbung vereiniges Verfahren fur die unter-
suchimg des plasmatischen zelleibs. Ber. Deui. Boi. Gesel., 1, hk-\7.
(Picro-nigrosin for chromatin.)

PlANESE, G.

1896. Beitrag zur Histologic und Aetiologie des Carcinoms. Beitrag zur
Path. Anat. u. AUgem. Path., Suppl. I, 193 pp. (Malachite green
and martins yellow with acid fuchsin.)

Phenani, a.

1902. Contribution a I'etude de la ciliation. Arch. (T Anat. Micro., 5. (Light
green with haematoxylin.)

Raniver.

1868. Technique microscopique. Arch. de. Phi/s. 1, Xo. 2, 319-321; No. 5,
666-670. (First use of picro-carmin in a single procedure.)

Rawitz, B.

1899. Bemerkungen uber Karminsaure und Hamatein. Anat. Anz., 15,
437-Ui.
REtiTER, Karl.

1901. Uber den farbenden Best andteil der Romanowsky-Xochtschen Malaria
plasmodien farbung, seine Reindarstellung und praktische Verwen-
dung. Centhl.f. Bakt. I Abt., 30, ^45-^-56'. (Dis.solves precipitate of
Nocht stain in absolute alcohol plus anilin oil.)
Robertson, O. H.

1917. The effects of experimental plethora on blood production. J. Exp.
Med., 26, 221-237. (Use of brilliant cresyl blue for staining reticu-
lated blood cells.)
Robinson, H. C. and Rettger, L. F.

1916. Studies on the use of brilliant green and a modified Endo's medium in
the isolation of Bacillus tNphosus from feces. ./. Med. Re.^. N. S.. 29,
363-376.

ROMANOV.SKI. D. L.

1891. On the question of parasitology and therapy of malaria (In Russian).

Imp. Med. Mililari/ Acad., ^ Dissert. No^ 38, St. Petersburg, 1891.

(Proposes combination of eosin and mthylene blue for staining blood.)
1891. Zur Frage der Parisitologie und Therapie der Malaria. St. Petersb. Med.

Wochenschr.. 16, 297-302, 307-315. A slightly condensed version of

the above.

Rothberger, C. J.

1898. Differential diagno.«tische unersuchungen mit gefarbten Xahrboden.
Centbl. f. Bakt. I Abt., 24, 513-518. (Neutral red as indicator in
media for differentiating typhoid and colon bacilli.)

SCHAFFER.

1888. Die Farberei zuni Studium der Knochenentwicklung. Zcit. Wis. Mikr.,
5, 1-10. (Fast yellow for bone; Congo red for embryo sections.)

SCHULTZ. G.

1923. Farbstofftabellen. 6 Aufl. Berlin. Bd. 1, 385 pp; Bd. 2, 290 pp.

Schwartz, E.

1867. Ueber cine Methode doppelter Farbung Mikroskopischer Objecte und
ihre Anwendung, etc. Sitz. berichte d. k. Acad. d. Wiss. Wien Bd. 55,
Eft. 1, 671-689. (First double staining.)

Schweiger-Seidel, F., and Dogiel, J.

1866. Ueber die peritoneal Hiille bei Froschen und ihren zusammenhang mit
dem Lymphagefassysteme. Ber. d. k. Sachs. Gessel. d. Wiss. zu
Leipzig, 18, 2Jt7-25If. (Use of carminates Avith acetic acid.)

143

Scott, R. E., and French, R. W.

1924a. Standardization of Biological Stains. Mil. Surg., Aug. 192Jf., 15 pj).
1924b. Standardization of Biological Stains. II Methylen blue. Mil. Sura.,

Sept. 192J,, 16 pp.
1924c. Standardization of Biological Stains. III. Eosin and haematoxylin.
Mil Surg., Nov. 1924 > 8 pp.
Smith, Louise.

1920. The hypobranchial apparatus of Spelerpes bislineatus. J. Morph., 33,

527-583. (Use of methylene blue in staining cartilege by the Van
Wijhe technic.)
Society of Dyers and Colourists.

1924. Colour Index. Edited by F. M. Rowe. Publi'ihed by the Society,
Bradford, Yorkshire, England.
Spulek, a.

1901. Ueber eine neue stiickfarbemethode. Deut. Med. Wchnschr., 2*7 y
Vereine-Beilage No. 14, 116. (Iron alum cocchineal.)
Stilling, H.

1886. Fragmente zur Pathologie der Milz. Virchows Arch. f. Path. Anal. u.
Physiol. 103, 15-88. (Iodine green for amyloid).
Teichmuller, W.

1899. Die eosinophile Bronchitis. Deut. Arch. Klin. Med., 63, -^^^-ffSS.
(Eosin for staining sputum, followed by methylene blue.)

TORREY, J. C.

1913. Brilliant green broth as a specific enrichment medium for the para-

t%'poid-interiditis group of bacteria. J. Inf. Dis., 13, 268-72.
Unna, p. G. '

1891. Ueber die Reifung unserer Farbstoffe. Zts. Wis. Mikr., 8, ^75-^7.
(Polychrome meth^dene blue).

1921. Chromolyse. Abaerhaldens Handb. der Biol. Arbeitsmethoden. Aht. 6,

Tcil 2, Hft. 1, {Liefng. 17) 1-62.
Vaughan, R. E.

1914. A method for the differential staining of fungus and host cells. .-Inn.

Mis. Bof. Gard. 1, 21^.1-21^2 . (An adaption for botanical purposes of
Pianese's staining malachine green acid fuchsin and martins yellow.)
Vinassa, E.

1891. Beitrage zur pharmakognostischen Miki'oskopie. Zts. Wis. Mikr., 8
(Auramin for plant sections.)
Waldeyer.

1 864. Untersuchungen iiber den Ursprung und den Verlauf des Axsencylinders
bei Wirbellosen und Wirbelthieren sowie iiber dessen Endverhalten
in der quergestreiften Muskelfaser. TIenle & Pfeifers Zeitschr. f.
rationelle. Med. 3 Reihe. Bd. 20, 193-256. (First attempt to stain with
logwood extract. Early use of anilin dj'cs).
Weigert, Carl.

1881. Zur Technik der mikroskopischeu Bakterien untersuchungen. Vir~
chow's Arch. f. Path. Anat. u. Phys., 84, p. 275-315. (Gentian violet
for fibrin and Neuroglia.)
1898. Ueber eine Methode zur Farbung elastischer Fasern. Centbl. f. Allgeni.
Path. Anat., 9, 289-292. (Fuchsin for elastic tissue).
Williams, B. G. R.

1923. Cresylecht violet, a rare dve. Jr. Lab. & Clin. Med., 8, No. J, Jan.

1923. i pp.
Wixogradsky.

1924. Sur Fetude microscopique du sol. C. R. Acad. d. Sci. 179, 367. (Ery-

throsin for staining bacteria in soil.)

ZiMMERMANN, A.

1893. Beitrage zur Morphologic und Physiologic der Pflanzenzelle Bd. IT.
Tubingen, 1893. 35 pp. (Iodine green as chromatin stain for plant
cells.)

ZSCOKKE, E.

1888. Ueber einige neue Farbstoffe beziiglich ihrer Verwendung zu histofo-
gischen Zwecken. Zts. Wis. Mikr., 5, .^65-^70. (Benzopurpia in
contrast to haematoxylin.)

144

REPORTS OF COMMISSION ON
STANDARDIZATION OF BIOLOGICAL STAINS

AND OF

RELATED COMMITTEES

Com. on Bact. Technic, of Soc. Amer. Bacteriologists, H. J. Couii, chairman.
1921. The Production of Biological Stains in America. Science, 53, 289-290.
1922a. An Investigation of American Stains. J. Bad., 7, 127-li8.
1922b. An Investigation of American Gentian Violets. J. Ba£t., 7, 529-536.
Com. on Standardization of Bioi.. Stains, of Nat. Res. Council, H. J. Conn,
chairman.
1922a. The Standardization of Biological Stains, Science, 55, |J-4i-

American Biological Stains compared with those of Griibler. Science,

55, 28^-285.
Preliminary Report on American Biological Stains. Science, 56,

156-160l
Collaborators in the Standardization of Biological Stains. Science,
56y 59Jt-596.
Commission on Standardization of Biol. Stains. H. J. Conn, Chairman.
1922.a The Present Supply- of Biological Stains. Science, 56, 562-563.
American Eosins. Science, 56, 689-690.
The preparation of Staining Solutions. Science. 57, 15-16.
Safranin and Methyl Green. Science, 57, 30^-305.
Thionin. J. Dairy Science, 6, 135-136.

Dye Solubility in Relation to Staining Solutions. Science, 57, 638-639.
Standardized Nomenclatures of Biological Stains. Science, 57, 638-639.
Certified Methylen Blue. Science, 58, 'il-/t2.
Investigations Concerning Imported Biological Stains. Science, 54,

328-331.
Certified Safranin. Science, 54, 556-557.
A report on Basic Fuchsin. Science, 60, 378-388.
Certified Stains— What Thev Are and How to Obtain Them. ./. Lab.

and Clin. Med., 10, 321-322.
New Applications of Biological Stains. J. Chein. Education, 2, 18'^-
185.

1922b.

1922.C
1922d.

1922b.

1923b.

1923c.

1923d.

1923e.

1923f.

1923g.

1924a.

1924b.
1924c.
1925a.

192ob.

145

INDEX

In this index the d^'es named are printed either in bold-faced type or italics;
preferred designations are in bold-faced type, synonyms in italics. Figures in
bold-faced type indicate the principal references.

Absorption curv^es, 28-31

Acid bordeaux, 35

Acid dyes, 16

Acid fuchsin, 22, 32, 64, 118, 134

Acid green, 62

Acid magenta, 64

Acid orange, 37

Acid rubin. 64

Acid yelloiv, 36, 41

Acid yellow D, 36

Alcohol soluble eosin, 79, 8U

Algae, .staining of, 57, 73, 81

Albert stain, 50, 115

Alizarin, 22, 42, 91

Alizarin carniin, 43

Alizarin No. 6, 43

Alizarin orange, 41

Alizarin jpurpurin, 43

Alizarin red, water sol., 43

Alizarin red S, 43, 1 1 3

Alizarin sulphate, 43

Alizarin yellow R, 41

Alizarin yellow GG, 41

Altmann, 65

Amaranth, 37, 112

Ambler, 52

Amethyst violet, 56

Amido-azins, 22, 53-55

Ammonium bases, 15

Ammonium picrate, 32

Amyloid, staining of, 66, 67, 71

Andrade indicator, 65, 119, 134

Anilin, 11, 14

Anilin blue, ale. sol., 71

Anilin blue, W. S., 22, 72, 121

Anilin dyes, first use of, 9

nature of, 1 1
Anilin red, 63
Anthracene yellow, 41
Anthracene yelloiD RN. 41
Apathy, 97, 127
Archelline 2B, So
Ascaris eggs, staining of, 61
Auramin, 22, 60, 117
Aurantia, 33, 110
Aurin, 74
Aurin R, 74
Auxochromes, 14, 15
Axis cylinders, staining of, 37, 40, 73
Azidine blue SB, 41
Azin radical, 17, 53

Azins, 22, 53-57
Azobenzene, 17
Azo-bordeaux, 35
Azo dyes, 17, ^1, 33-41

radical, 17
Azo rubin, 37
Azure I, 47, 48
Azure II, 49, 89
Azure A, 48, 90
Azure B, 48

Bacillus subtilis, 135

Bacteria, staining of, 46, 62. 68, 76,

81, 83
Bacteriostatic action of gentian violet,

68, 69, 135
Balch, 89
Basic dves, 15

Basic fuchsin, 22, 28, 63, 118, 1.33
Basic rubin, 63
Benda, 43, 5Q, 62, 68. 97, 98, 113. 116.

117, 119, 128
V. Beneden, 61, 1 17
JocncKG 0
Ben.slev, 35, 65, 70, 98, 111, 119, 128,

134
Bensley-C'owdrv technic, 65, 70, 119,

120
Bensley's neutral gentian, 34
Benzamine blue 3B, 41
Benzene, 11
Benzene yellow, 41
Benzene yellow PN, 41
Benzo blue SB, 41
Benzoin sky blue, 41
Benzopurpin 4B, 40, 113
Bergonzini, 36, 111
Berlin blue, 72
Bernthsen, 48, 49, 50, 56, 88
Best, 93

Biebrich scarlet, water sol., 39, 112
Bindschedler's green, 44
Bismarck brown G, R and GOOD, 39
Bismarck brown Y 21, 39, 112
Blood stains, 47, 49, 50, 54, Qo, 70, 71,

88-90
Bohmer, 9, 97, 127
Bone, staining sections of, 36
Bordeaux, 37

Bordeaux B, BL, G, and R, 35
Bordeaux SF, 37

146

Bordeaux red, 35, 111
Hbttcher, 1)
Brazalum, 96
Brazil, 117
wood, 9.)
Brazilein, 96
Brazilin, 95, 126
Brilliant blue C, 51
Brilliant cresyl blue, '-22, 51, 115
Brilliant green, 22, 61, 117
Brilliant pink\ 77
Brom cresol green, 85
Brom cresol purple, 85
Brom chlor phenol blue, 85
Brom phenol blue, 85
Brora phenol red, 85
Brom thymol blue, 85
Broun salt R, -il
Biitschli, 128

Caesar red, 80

Calcium salts, detection of, 43

dauary yellow, GO

Cancer tissue, staining of, 32, 61, do

Capri blue, 53

Carbinol, 19, 59

Cardinal red, 41

Carmin, 7, 8, 92, 124

Carminic acid, 93, 125

Carmin naphtha, 41

Cartilage of frogs, staining of, 51

Cellulose, staining of, 40, 62. 65

Cerasin, 35, 41

Cerasin red, 38

Cerotin orayige, 41

Chamberlain, 57, 81, 82, 116

Champy-Kull technic, 33, 110

China blue, 72

Chlor azol blue 3B, 41

Chlor phenol red, 85

Chromatin, staining of, 50, 56, 67, 70,

71, 93, 97
Chrom black, 41
Chromogens, 14, 15
Chromolysis, 36, 54, 57, 70, 73, 94, 103
Chromophores, 14, 15, 16-18
Chrom violet, 74
Chrysoin, 41
Chrysoidin Y, 41
Chrysoidin R, 41
Ciaccio, 71, 120
Cocchlneal, 92, 123
Cohn, 8, 93
Color, relation to chemical formula, 20

relation to light absorption, 25
Colour Index, 23
Congo, 40
Congo blue 3B, 41
Congo red, 21, 40, 112
Congo shy blue, 41

Conn, 68, 123

Connective tissue, staining of, 32, 34,

65
Copper, detection of, 97
Corallin red, 22, 74
Corallin yellow, 74
Cortex, staining of, 65
Corti, 8, 93
Cotton blue, 72
Cotton orange, 41
Cotton red, 40
Cotton red 4B, 40
Cowdrv, 35, 65, 119, 120
Cresyl blue 2RN and BBS, 51
Cresyl echt violet, 52
Cresyl red, 85
Cresyl violet, 52, 115
Croceinc scarlet, 39
Crystal ponceau 6R, 41
Crsytal violet, 21, 28, 67, 119, 134
Cutinized tissues, staining of, 56
Czokor, 92, 123

Daddi, 38, 112

Dahlia, Qo

Dahlia B, 66

Dark brown salt /?, 41

Delafield, 80, 97, 127, 137

Diamin red JfB, 40

Diamond black F, 41

Diamond f la vine, 41

Diamond fuchsin, 63

Diamond green, 61

Dianil blue H3G, 41

Dianil blue HOG, 41

Dianil red ^C, 40

Dianthin B, and G, 81

Diazin green, 35

Differentiation, first use of, 9

Di-phenyl methane dyes, 60

Diphtheria organism, staining of, 47,

132
Direct red, 40
Direct red 4B, 40
Dogiel, 9
Double green, 69
Double scarlet, 39
Double staining, first use of, 9
Dubreuil, 72, 121
Duesberg, 68
Dye, definition of, 15

indexes, 23
Dyes, classification of, 20

nomenclature of, 22

solubilities of, 24

spectrophotometric analysis of, 25-
31

Ebbinghaus, 36, 111
Ehrenberg, 7, 93

147

Ehrlich, 34, 36, 37, 52, 56, 57, 65, 66,

70, 76, 77, 87. 88, 90, 07, 110, 120,

121, 127
Ehrlich-Biondi-Heidenhain stain. 34,

36, 65, 70, 111, 118, 120
Elastic tissue, 57, 62, 94
Einbrvos, staining sections of. 35. 40,

54, 72, 95
Emerald green, 61
Endo medium, 62, 63, 118, 134
Eosin, alcoJiol soluble, 79, 80
Eosin bluish, 80

Eosiii BN, B, BW, and 7) AT, 80
Eosin J, 81
Eosin S, 80

Eosin scarlet B and BB, 80
Eosin W or WS {i. e. wafer soluble). 79
Eosin Y, 79. 122, 136
Eosinophile granules, 79
Epithelium, staining sections of. 73
Erythrocytes, 61
Erythrosin, 22, 122, 81
Erythrosin, B. R, and G, 81
Erythrosin BB, 82
Excelsior broicn, 39
Ethyl eosin, 80
Ethyl green, 61
Extinction coefficient, 27 '
Eurhodins. 53-55

Earis, 35, 54, 111, 115

Vast acid green N, 62

East blue 3R, 53

East red, 37

East red A or Ai', 41

Fast red B or P, 35

East red 0, 41

Fast yellow, 35, 111

Eat stains, 34, 38, 52

Fettponceaii, 38

Fibrin, staining of, 68

Eischel, 60, 117

Elemming, 9, 57, 68

Elemming triple stain, 34, 56, 67, 102,

110, 116, 119, 135, 136
Eluorane, 78

derivatiyes, 22, 78-83
Fluorescein, 78
Foot, 112
French, 47, 48
Erey, 9

Frogs, staining cartilage of, 51
Frost, 46, 114, 137
Frozen sections, 67, 137
Fuchsin, acid, 16, 22, 32, 64, 118, 134
Fuchsin, basic, 9, 16, 19, 22, 28. 63,

118, 133
Fuchsin NB, 64

Fuchsin S, SN, SS, ST, or .S77/, 64
Fuchsinophile granules, 62

Fungi, staining in sections of infected
plants, 33

Galeotti, 70, 120

Galli, 73

Gentian blue, 71

Gentian violet, 22. 66, 67, 68, 119, 134

Gerlach, 8, 9

Giemsa, 49, 89, 114

Gierke, 7

Gold orange, 36, 37

Gold yellow, 41

Gonococcus, staining of, 54, 76, 70

Goppert, 8, 93

Graberg, 35, 111, 114

Gram stain for bacteria, 39, 56, 68, 76,

100, 101, 119, 135
Gray R, B, BB, 57
Grenadier, 93, 94. 124
Griesbach, 37. 40. 77, 112, 113, 120,

121
(iriibler, 5, 89

Ilaemahim, 97

Haematein, 96, 126

Haematoxylin, 9, 96, 126, 136

Hansen, 92, 124, 127, 128

Hartig, 8, 93

Heidenhain, 35, 97, 127, 128, 137

Held, 81, 122

Ilelianthin, 3(5

Heliotrope B, 5G

Helvetia blue, 72

Henneguy, 127

Hermann, 9

Hexamethyl violet, 67

Hickson, 96, 126

Hoffman green, 71

Hoffman violet, 21, 65, 119

Holmes, 52

Hoyer, 124

Huber, 73, 121

Hucker, 68, 112, 116, 119

Imperial yellow, 33

Indamin radical, 17

Indamins, 22, 44

Indicators, 36, 40, 42, 43, 54, 65, 73,

83-5, 95
Indigo, 8, 91
Indigo blue, 91
Indigo-carmin, 92, 123
Indigotinc la, 92
Indin blue 2RD, 53
Indulin black, 57
Indulins, 22, 57
Insects, staining tissue of, 46
lodo-eosin B and G, 81
Iodine green, 71, 120
Iodine violet, 65

148

Iris violet, ofi
Iron, detection nl". '.)
Jsoruhin, 64
/srael, 9.5, V2Q

Janus green B, 35, 1 II
Janus red 41
Jarotsky, 57, 116
Jenner, 89
Juergens, G(i, 119

Kaiser, 128
Kehrraann, 48
Keratin, staining of, 36
Klebs, 40, 113
Krumwiede, 61, 117
Kiiltschitzky. 57, 116

Lactone ring, 78

Langerhans, islands of, 34

Lauttis violet, 45

Lazarus, 56

Leather brown, 39

Lefas, 71, 120

Leishman, 89

Leuco-base, 19, 59

Leuco compounds, 18-20

Leuco-fuchsin, 19, 62

Levine, 47, 114, 136

Light green, SF vellowish, 22, 62, 69,

117
Light green 2G, 3G, J^G, or 2GN, 62
Light green N, 61

Lignified tissue, staining of, 50, 56, 68
List, 79, 122

"Little plate" teclmic, 46, 137
Litmus, 95
Loeffler, 48
Logwood, 9, 95

Lorrain Smith fat stain, 52, 115
Lyons blue. 71

Maas, 61, 117

MacNeal, 48, 49, 89, 90

Magdala red, 22, 56, 81, 82, 116

Magenta, 63

Malachite green, 22, 32, 61, 117

Malachite green G, 61

Mallory, 79, 82, 89, 98, 105, 110, 114,

119, 122
Mallory connective tissue stain, 34, 65,

73, 110, 118, 121, 134
Manchester broum, 39
Manchester yelloic, 32
Mandarin G, 37
Mann, 7, 72, 79, 121, 122
Marine blue, 72
Martius yellow, 32, 110
Maschke, 8
Mast cells, staining of, 66, 70, 76

Mauveine, 9

Mayer, 92, 93, 94, 96, 97, HS, li24,

125, 126; 127
McClung, 98
Meldolas blue, 53
Methyl blue, 72, 121
Methyl eosin, 79
Methyl green, 21, 22, 69, 120
Methyl orange, 21, 36, 111
Methyl violet, 21, 22, 66
Methyl violet lOB, 67
Methylene azure, 47, 48, 88, 89, 114
Methylene blue, 22, 30, 46, 114, 132
Methylene blue NN, 51
Methylene blue 0, 50
Methylene green, 50, 114
Methylene violet, 47, 49, 88
Methylene violet RRA, 56
Meves. 68

Michaelis, 35, 38, 111, 112
Milk, staining of, 132
Mitochondria, staining of, 33, 35, 43,

65, 68, 70, 98, 134
Mitosis, 98
Moll, 95, 126
Mucin, staining of, 46, 71
Miiller, 8, 32, 110, 117, 119
Muscle fibers, staining of, 65, 92, 98

Naphthaline pink, 5G

Naphthaline red, 56

Naphthamine blue, 41

Napthamine blue 3BX, 41

Naphthol blue, 53

Naphthol orange, 36

Naphthol red, 37

Naphthol yellow, 32

N aphthylamine pink, 56

Narcein, 37, 111

Nervous tissue, staining of, 37, 40, 43,

47, 52, 57, 62, 72, 81, 98
Neuroglia, staining of, 68
Neutral gentian (Bensley), 34
Neutral red, 22, 54, 115
Neutral violet, 54, 115
Neutral stains, 76, 77, 79, 87-90
New blue B, 53
New f uchsin, 20, 64
New methylene blue N, 51
New pink, 82
New Victoria green, 61
Niagara blue 4B, 41
Niagara blue 3B, 41
Niagara sky blue, 41
Night blue, 71

Nigrosin, water sol., 22, 57, 116
Nile blue A, 52

Nile blue sulphate, 22, 52, 115
Nissl granules, staining of, 54

149

Xitro dyes, 21, 32

radical, 18
Nocht, 88, 89
Nopalin G, 80

Oil red, 38

Oil red IV. 38

Oil yelloic, 41

Orange I, 36

Orange II, 37

Orange III, 30

Orange IV, 36

Orange A, P, or R, 37

Orange extra, 37

Orange G, 21, 28, 34, 110, 136

Orange GO, or GMP, 34

Orange N, 36

Orange 7?, 41

Orcein, 94, 126

Ortho-sulpho-benzoic acid, 83

Oxazins, 22, 51-53

Oxyphile granules, 79

Oxyquinone dyes, 22, 42

Paladino, 39, 112

Pancreatic tissue, staining of, 57

Pappenheim, 50, 70, 76, 90, 115, 120,
121, 135

Para-fuchsin, 63

Para-magenta, 63

Pararosanilin, 18, 20, 60, 63

Pararosolic acid, 74

Paris blue, 71

Paris violet, 66

Partsch, 92, 123

Pelagetti, 112, 123

Perkin, 9

Petroff, 61, 117

Pfitzer, 57, 116

Phenazin, 17, 5t}

Phenol, 15

Phenol red, 84

Phenolphthalein, 22, 83-4

Pheneylene blue, 53

Phenylene brown, 39

Phenyl-methane dyes, 22, 58-74

Phloxine, 82, 122 '

Phthalic acid, 83

Phthalic anhydride, 77, 83

Pianese, 32, 61, Q5, 110, 117, 118

Picric acid, 9, 15, 21, 32, 110

I'icro-carmin, 9

Pith, staining of, 65

Plants, fungus diseases of, 33, 61, 65

staining sections of, 60
• Plgsma fibrils in epithelium, 94

Platelets, staining of, 67 •

Ponceau B^ 39
. Ponceau 3B, 38

Ponceau 6t{, 41

Protein, staining of, 56, 70

Protozoa, staining of, 70

Pseudo-base, 19

Purpurin, 43, 113

Pyoktanin blue, 66

Pyoktanin yellow, 60

Pyoktatiimum aureum, 00

Py renin B or G, 22, 76, 121, 135, 136

Pyronins, 22, 75-6

Pyrosin B and ./, 81

Quinoid ring, 13, 17, 18, 42, 45, 59. 65,

73, 84
Quinone, 13
Quinone-imide dyes, 22, 44-57

Ranvier, 9, 124

Rawitz, 94, 125

Red violet, 65

Renter, 89

Rhodamine B, 22, 77, 121

Rhodaminc 0, 77

Rhodamine S, 77

Rhodamines, 22, 77-8

Rosanilin, 20, 63

Rosanilins, 62-73

Robertson, 115

Roccellin, 41

Romanovskv, 79, 87, 88, 89, 114, 122

Rose Bengal, 22, 83, 123

Rosen, 77

Rosolic acids, 22, 73-74

Rothberger, 115

Rubin, 20, 03

Rnbidin, 41

Safranin O, 22, 55, 115, 133

Safranins, 17, 22, 54-57

Safrosin, 80

Salamander larvae, staining of, 60

Salicin black, 41

Scarlet B, or FX\ 39

Scarlet G, or B, 38

Scarlet J, J J or V, 80

Scarlet red, 38

Schaffer, 36, 40, 111, 113

Scharlach B, 41

Scharlach R, 38

Schneider, 93, 124

Scott, 47, 48

Schr otter, 43, 113

Schultz, 127

Schultz' Index, 23, 106, 107, 109

Schwa rz, 9

Schweigger-Seidel, 8

Silver Gray, 57

Skin, staining sections of, 36

Smith, Lorrain, 52, 115

Smith, Louise, 51

Society of Dyers and C'olourists, 23

Solid green, 61

150

Soluble blue 3M or 2R. 72
Spectrophotometer^ 26

diagram of, 27
Spectrum, 25

diagram of, 26
Spermatozoa, staining of, 62
Spirit blue, 71
Spore coats, staining of, 56
Spuler, 92, 123
Sputum, staining of, 79
Steel gray, 51
Strobell, 112
Stroebe, 73, 121
Succineins, 77
Sudan III, 21, 34, 38, 112
Sudan IV, 21, 38, 112
Sudan S, 41
Sudan G, 38
Suda7i red, 56
Sulfonic dyes, 16
Sulphonphthaleins, 22, 84-5
Sultan J,B, 40
Swiss blue, 46

Tadpoles, vital staining of.

Teichmiiller, 79, 122

Thiazins, 22, 44-51

Thiersch, 8

Thionin, 22, 30, 31, 45, 113, 137

Thymol blue, 85

Tony red, 38

1'oluene, 14, 58

Toluidinc, 14

Toluldine blue 0, 50

Toluylene blue, 44

''Triacid mixture" (Khrlich), 34, 87, 88

Tri-j)henyl methane dyes, 22, 60-74

Triple stain (Flemming), 34, 5(y, 67,

102, 110, 119, 135, 136
Trinitrobenzene, 15
TropaeoUn D, G, 00. and 000, 36
Tropaeolin 000 Xo. ?, 37
Tropaeolin 0, 41

Tropaeolin Y, 41
Trypan blue, 41, 113
Trypan red, 40, 113

Tubercle organism, staining of, 62
Tumor tissue, staining of, 46, 52, 98
Typhoid organism, staining of, 61, 62

Unna, 36, 39, 54, 57, (i6, 70, 73, 78,
82,89,94, 103, 104, 111, 115, 116,
121,122, 126

Van Gieson, 32, 65, 98, 110, 118, 128

A'an Wijhe, 51, 124

Vaughan, 110, 117, 119

Vesuvin, 39

Victoria rubin, 37

Vinassa, 60. 117

Violet C, G, or 7B, OT

Violet R, RR, or JfRX, lio

Vital staining, 35, J^7, 52, 54, 60

Waldeyer, 9

Water blue, 72

Wafer soluble eosin, 79

Weigert, 67, 98, 118, 119, 125, 127, 128

V. Wellheim, 125

Williams, 52, 115

Winogradsky, 81, 122

Wood, staining of, 50, 56, 68

Wool orange 2G, 34

Wool red, 37

Wright stain, 87, 89, 136

Xantheue dyes, 22, 75-85
Xylem, staining of, 70, 71
Xylene, 14

Zacharias, 125

Ziehl-Neelson method, 62, 118. 133
Zimmermann. 71, 120
Zschokke, 41, 113

151