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STAIN TECHNIQUE

BY

W. H, EMIG

University of Pittsburgh

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1941

Copyright, 1941 by
W. H. Emig

Printed in the United States of America

THE SCIENCE PRESS PRINTING COMPANY
LANCASTER, PENNSYLVANIA

PREFACE

Stain Technique is designed to provide concise instruction in
the correct usage of biological stains by introducing the chemistry
of color precipitates, the arrangement of stains into a few classes
depending on their manner of application, and the outline for stain-
ing with combinations of two, three, or four dyes. The results of
numerous tests with each dye indicate the action of acids, alkalies,
and salts; the composition of effective stains; the influence of re-
agents on the fastness of color; the affinity for tissues of algae,
fungi, mosses, vascular plants, invertebrate and vertebrate ani-
mals; and the combination of stains which provide contrasting
colors. Because most of the experimental products are of nega-
tive value, many of the dyes merit only a brief statement. A few
superior and distinctive coal-tar dyes are worthy of admission into
standard methods. Hence, the fundamental plan of this textbook
is extended to include those phases which assist the student in a
practical application of stain technique.

ACKNOWLEDGMENTS

During the progress of my investigations on microtechnique,
I received helpful suggestions from my colleagues, Professors
A. B. Wallgren, Alexander Lowy, and J. K. Miller. Dr. Russell
Gottschall and Dr. Primitivo Colombo made many of the prelimin-
ary tests on coal-tar dyes. Dr. Anna Mary Carpenter ably assisted
in a number of ways, especially in the preparation of the manu-
script. I am greatly indebted to the several directors of the tech-
nological laboratories of both E. I. du Pont de Nemours and Com-
pany, Wilmington, Del., and the National Aniline and Chemical
Company, Philadelphia, Pa., for data on color technique and for
samples of mordant, sulfur, and vat dyes.

William H. Emig

University of Pittsburgh

March 31, 1941

CONTENTS

PREFACE 3

ACKNOWLEDGMENT 4

I. DYES FIRST USED 7

II. CHEMICAL AND PHYSICAL PROPERTIES OF

DYES 9

Color Precipitates

Ferric ferrocyanide

Ferric tannate

Hematein
Reversible Actions
Oxidation
Reduction
Physical Properties

III. GENERAL USES OF BIOLOGICAL STAINS 13

Acid Dyes
Direct Dyes
Mordant Dyes
'Basic Dyes
Nomenclature of Dyes

IV. STAINING WITH CONTRAST DYES 18

Selecting a Combination of Stains
Combinations of Two Stains

Crocein Scarlet MOO and Niagara Sky Blue
Two Dyes Used Separately
Two Dye-solutions Mixed
Sun Yellow and New Methylene Blue
Rhodamine 6G and Aniline Blue
Combinations of Three Stains
Spot Test Using Basic Dyes
Time Test Using Acid Dyes
Combinations of Four Stains

V. BIOLOGICAL STAINS 25

A. Synthetic Organic Dyes

C. I. Nos.

Class I. Nitroso Colouring Matters 5

Class II. Nitro Colouring Matters 7-10

Class III. Monoazo Colouring Matters 16-211

Class IV. Disazo Colouring Matters 248-520

Class V. Trisazo Colouring Matters 581-594

Class VI. Tetrakisazo Colouring Matters ... —

•5

53677

6 W. H. E M I G

VI. BIOLOGICAL STAINS 44

A. Synthetic Organic Dyes (Continued)

Class VII. Stilbene Colouring Matters 620-622

Class VIII. Pyrazolone Colouring Matters .... 640
Class IX. Ketonimine Colouring Matters .... 655
Class X. Triphenylemethane and Diphenyl-
naphthylmethane Colouring Mat-
ters 657-736

Class XL Xanthene Colouring Matters 739-783

Class XII. Acridine Colouring Matters 785-793

Class XIII. Quinoline Colouring Matters 801-806

Class XIV. Thiazole and Thiobenzenyl Colour-
ing Matters 812

Class XV. Indamine Colouring Matters —

Class XVI. Indophenol Colouring Matters .... —

Class XVII. Azine Colouring Matters 825-865

Class XVIII. Aniline Black and Allied Colouring

Matters —

Class XIX. Oxazine Colouring Matters 876-917

Class XX. Thiazine Colouring Matters 920-931

Class XXI. Sulfur and Sulfide Colouring Mat-
ters 948-1012

Class XXII. Hydroxyketone Colouring Matters. —
Class XXIII. Anthraquinone Colouring Matters . 1027-1067
Class XXIV. Anthraquinone Vat Colouring Mat-
ters 1101-1162

Class XXV. Arylidoquinone Colouring Matters. —

Class XXVI. Indigoid Colouring Matters 1177-1180

VII. BIOLOGICAL STAINS 66

B. Natural Organic Dyestuffs 1239-1249a

C. Inorganic Colouring Matters 1288

Index 72

o

Chapter I
DYES FIRST USED

During the early investigations of dissected tissues, different
chemicals were applied in the hope of finding some means by which
the structural details of cells could be seen more clearly under a
lens. Frequently, mineral acids were tried; a tincture of iodine
was a common reagent. Iodine is still used in the examination of
temporary mounts where it colors proteins brown and starches
blue. The first organic colors tested as biological stains were the
mordant dyes extracted from insects, shellfish, roots, leaves, and
wood. The technique inherent in the application of these natural
dyes developed slowly because the need and action of mordants was
little understood. With the exception of Carmine and Haema-
toxylin the natural dyes have been replaced in microtechnique by
synthetic coal-tar dyes.

Occasionally, coal-tar dyes which are important in a special
branch of commercial work are tried in histological procedures.
If the results prove satisfactory to the investigator, the dye may be
introduced as a special laboratory technique; then the dye is con-
sidered a biological stain. The biologist, however, can learn many
important facts from an examination of the technique which has
been continuously improved by industrial chemists. Large-scale
dyeing of wool, silk, and cotton has demonstrated the values and
limitations of dyes. Consequently, the manufacturer insists both
on a selection of a dye appropriate to each fabric and on a technique
that has proven universally effective.

Coloring matters, according to their manner of application are
designated as acid, direct, sulfur, mordant, vat, or basic dyes.
Acid dyes are used on wool fabrics in a bath containing sulfuric,
acetic, or formic acid. Because of their ability to stain cotton
without an intervening agent, a few dyes are named direct dyes.
On cotton fabrics, sulfur dyes are resistant to the injurious action
of alkalies. Naturally occurring dyes require the aid of a mordant
including compounds of aluminum, antimony, chromium, iron, tin,

7

8 W. H. EMIG

or copper. Frequently, tin salts are employed with Cochineal.
For light colors, an aluminum salt is preferred because it does not
alter the tint. Chromium and iron compounds, which have a
deadening effect on bright tints, are combined with dark colors
such as Haematoxylin.

Vat dyes on cotton cloth are very fast to light. They are
worked into a paste with a strong alkaline liquor and are reduced
to a soluble form with sodium hydrosulfite; this caustic solution is
called a vat. Basic dyes provide brilliant colors either on silk, in
a weak acid solution, or on cotton with the aid of tannic acid. The
latter method of operation suggested the term tannin dyes.

Chapter II
CHEMICAL AND PHYSICAL PROPERTIES OF DYES

Color Precipitates. A discussion of the actions involved in
the process of staining may be introduced with examples of color
precipitates. A few colloidal pigments give a tissue the same ap-
pearance as if a coal-tar dye were used. A blue-black stain such
as the tannate of iron is obtained by applying separately either
tannic acid and ferrous sulfate, tannic acid and ferric chloride, gal-
lic acid and ferric chloride, or gallic acid and ferric ammonium sul-
fate. In like manner copper sulfate and potassium ferrocyanide
yield brown but ferric salts and potassium ferrocyanide give blue
precipitates. It is only through an exchange of radicals between
two salts that blue, brown, or black precipitates are produced. A
study of the conditions which favor the formation of a permanent
pigment in a test tube gives a clue to the correct procedure needed
for the application of many biological stains.

Ferric ferrocyanide. A brilliant blue precipitate is formed by
mixing equal quantities of 1 per cent ferric ammonium sulfate and
3 per cent potassium ferrocyanide. In the process of staining, the
material remains in a solution of potassium ferrocyanide for 15
minutes. After a rinse in distilled water, the tissue is covered with
a solution of ferric alum. With the precipitation of ferric ferro-
cyanide the tissue becomes blue ; the color is intensified by repeat-
edly alternating potassium ferrocyanide with ferric ammonium sul-
fate because the greater the amount of pigment, the darker is the
shade. Prussian Blue is suitable for either glycerin or balsam
mounts.

Ferric tannate. The addition of 1 per cent ferric ammonium
sulfate to 5 per cent tannic acid yields a blue-black, Hematein-like
pigment. An object left in tannic acid for 15 minutes, rinsed
with distilled water, and treated with ferric alum becomes blue-
black. A repetition of the action with tannic acid and ferric alum
increases the amount of ferric tannate.

Hematein. If a drop of ferric alum is added to an aqueous

10 W. H. E M I G

solution of Haematoxylin, a blue-black precipitate appears. This
pigment, Hematein, is the oxidized substance seen in tissues stained
with Haematoxylin. In staining, the section is placed first in a
solution of ferric alum, whereupon certain types of proteins absorb
the iron salt. A rinse in distilled water removes the ferric alum
from the surface of the tissue. Now, those parts that have re-
tained the mordant are colored in Haematoxylin ; other parts from
which the mordant is absent remain colorless.

Reversible actions. In the production of color precipitates,
the action remains more or less incomplete because the reverse ac-
tion also takes place under the same conditions. Since the extent
to which the precipitate is affected depends on the concentration of
the original salts, it is possible to adjust the conditions in staining
and thereby regulate the process in one direction.

With the formation of ferric f errocyanide, some of the precipi-
tate is dissolved in the presence of an excess of potassium ferro-
cyanide giving either a suspension of the precipitate or only a
colored solution. On the other hand, with a slight excess of ferric
ammonium sulfate, the pigment is not dissolved to an appreciable
extent.

A tissue left in tannic acid for 15 minutes, rinsed with distilled
water, and treated with ferric alum becomes blue-black. By re-
versing the order of application, using ferric alum first and then
tannic acid, some of the iron tannate dissolves in the tannic acid so
that within 15 minutes only a part of the original precipitate re-
mains. This Hematein-like pigment is very soluble in tannic acid
but less so in ferric alum.

A precipitate forms immediately after a drop of Haematoxylin
is added to 1 per cent ferric ammonium sulfate, but the Hematein
soon dissolves in ferric alum. In Heidenhain's method of stain-
ing, ferric ammonium sulfate is needed first as a mordant. After
a rinse in distilled water the tissue is overstained in Haematoxylin ;
the excess pigment is removed with ferric alum. During the pro-
cess of destaining the material must be observed under a micro-
scope. As soon as the correct degree of color has been attained,
the section is taken out of the ferric alum, but the reversible action

STAIN TECHNIQUE 11

continues until the section is washed with distilled water. After a
second rinse, in distilled water containing a trace of ammonium
hydroxide, the oxidation of Hematein is completed and the object
is stained deep blue-black. If the material is washed with tap
water containing sulfuric acid, the pigment is greenish black.

Oxidation. In microtechnique, the study of the conditions
which effect color is especially important ; the behavior of biological
stains differs with the physical and chemical properties of the
tissues and with the presence of chemical assistants. Many coal-
tar colors are more fast if they are developed by means of oxida-
tion. The importance of this reaction can be demonstrated with
a number of azo dyes which do not stain plant tissues unless an
oxidizing agent is added to the dye bath. If Erythrosin, a Xan-
thene dye, is absorbed by a tissue in the presence of potassium di-
chromate, the pink is changed to scarlet. With many of the coal-
tar dyes, however, the oxidized color is soluble in water or alcohol ;
consequently such stains are unsuited for permanent mounts.

In aqueous solutions of Picric Acid, plant and animal tissues
remain pale yellow. With the addition of acetic acid to the stain,
the yellow is increased but fades after five hours exposure to sun-
light. With the aid of potassium dichromate and acetic acid, the
color changes very little after more than 50 hours exposure to sun-
light. Thus, the presence of appropriate chemical assistants strik-
ingly modifies the fastness of Picric Acid.

Reduction. Dyes may be affected by reduction in a number
of ways. In a glycerin mount, ferric ferrocyanide fades to white
ferrous ferrocyanide unless a trace of potassium dichromate is
added to the dilute glycerin during dehydration. Most of the sul-
fur, mordant, and vat dyes insoluble in water or alcohol are
brought into solution by reduction. After the tissue has retained
the stain in its soluble state, the color is developed by re-oxidation.
The oxygen necessary to complete the action may be absorbed from
the air. • Hydrochloric acid in acid-alcohol changes many basic dyes
to the leuco compound, the form of dye which is not necessarily
colorless but which is usually more fugitive. For this reason, the

12 W. H. E M I G

destaining of basic dyes with acid-alcohol has been omitted from
the technique described in this text.

Physical properties. In a few staining procedures the ab-
sorption of color is entirely physical. If sections of germinating
corn grains remain in a solution of Sudan IV, the tissue containing
fats stains a bright red. The dye is absorbed by the fats but not
by the proteins or carbohydrates. Here, the oil soluble dye, un-
changed chemically, is held in suspension. The dye is extracted
from the fatty tissue as soon as the stained sections are changed to
strong alcohol, acetone, or xylene. In a similar manner, a dye dis-
solved in an oil such as clove oil or aniline oil is retained by a tissue
in the same form as it occurs in the oil suspension. Colors pro-
duced in this manner are not oxidized pigments; they disappear
gradually or dissolve in balsam.

Chapter III
GENERAL USES OF BIOLOGICAL STAINS

Biological stains are prepared from powdered dyes. Solution
is effected in water or dilute alcohol with the addition of chemical
assistants. In commercial dyeing certain chemicals are used both
as levelling agents to develop an even shade and as assistants to
exhaust the dye bath or to fix the dye to the fabric. Any sub-
stance added to a biological stain for the purpose of producing a
better differentiation of color may be considered a chemical assis-
tant.

A trace of sulfuric acid is added to acid dyes to increase the
staining action, provided precipitation does not occur; with basic
dyes acetic acid retards the staining process. Should a dye be pre-
cipitated by sulfuric acid, the effective sulfate ion is obtained from
sodium sulfate and the acid reaction from a weaker acid such as
acetic or formic acid. Whenever the dye is sensitive to all acids,
the oxidizing agent potassium dichromate is tried. Ammonium
alum is added to basic and direct dyes because the color becomes
more fast to washing. The sulfur and vat dyes are insoluble in
dilute acids but soluble in weak alkalies.

Chemicals necessary in the formation of color precipitates are
seldom added to the stain but are applied in separate solutions.
The salts of heavy metals especially aluminum, iron, and chromium
combine with mordant dyes to give insoluble color lakes. The
need for a chemical assistant is more clearly demonstrated with a
mordant dye than with a basic, acid, or direct dye.

The active biological stains are separated into two large classes
acid dyes and basic dyes. The basic dyes form a homogeneous
group; one type formula is sufficient for the stains used on either
whole mounts or paraffin sections. Although the acid dyes pro-
duce similar results on a particular tissue they differ in their man-
ner of application. These differences are indicated in the subdivi-
sions acid, direct, sulfur, mordant, and vat dyes.

13

14 W. H. E M I G

Acid dyes, Colour Index Numbers.

151. Orange II.

252. Crocein Scarlet MOO.

280. Biebrich Scarlet.

286. Crocein Scarlet 7B.

692. Acid Fuchsine.

707. Aniline Blue.

Many of the acid dyes are sodium salts of a sulfonic acid. The
principal salt-forming radical is the hydroxyl group. The radical
- S0 3 Na, which is not considered an auxochrome, renders the dye
more soluble in water and more acidic. If possible, 0.01 per cent
sulfuric acid (1 ml. of 1 per cent) should be added to each 100 ml.
of the dye solution. A few dyes are precipitated by sulfuric acid,
whereupon either acetic acid, formic acid, sodium sulfate, or potas-
sium dichromate becomes the chemical assistant. With many of
these dyes the amount of ionic dissociation of the molecule in water
is so slight that the stain is relatively inactive. The intensity of
the stain is increased by the presence of an acid, decreased with an
alkali.

The routine procedure with an unfamiliar acid dye requires
three tests. The first object taken from the stain at the end of 15
minutes is dehydrated in alcohol and cleared in xylene. An exami-
nation of the finished product shows whether or not a 15-minute
interval is sufficient. If the color is too light, the second section
is tested at the end of an hour. The third section, in turn, remains
in the stain 12 to 24 hours. The activity of acid dyes does not
extend beyond 24 hours. Naturally, if the first test at the end of
15 minutes gives too intense a color a shorter interval is indicated.

Acid dyes are especially suited for proteins, either in plant or
animal tissues. Within plants the proteins are more abundant in
parenchyma, cambium, phloem, and immature spores. It is impos-
sible to give a definite time limit for any particular dye because the
time differs both with the kind of tissue and with the fixative.
Orange II is sometimes too weak to color mature parenchyma in 24
hours, yet the meristem of root tips fixed in sublimate-dichromate-
acetic is deeply stained within a few seconds. On animal tissues,
Orange II requires an average of three to five minutes although
sections may be found that are not overstained in 24 hours.

STAIN TECHNIQUE 15

Direct dyes, Colour Index Numbers.

327. Direct Fast Scarlet 4BS.

370. Congo Red.

518. Niagara Sky Blue 6B.

520. Niagara Sky Blue.

620. Sun Yellow.

957. Sulfur Brilliant Blue.
1006. Sulfur Green.
1012. Sulfur Bordeaux.

The principal radicals of direct dyes are - NH 2 and - OH, the
auxochromes that characterize the basic and acid dyes respectively.
A direct dye is applied in neutral solutions containing sodium sul-
fate, in ^ per cent acetic acid, or in alkaline solutions. Direct dyes
containing alkalies cannot be used on paraffin sections but they are
often valuable for the coloring of minute objects mounted entire in
glycerin. Solutions of Direct Fast Scarlet 4BS, Niagara Sky
Blue, and Sun Yellow should contain both acetic acid and sodium
sulfate as chemical assistants. Because Congo Red changes from
red to blue in the presence of an acid, only sodium sulfate is used
in the stain. The direct dyes provide contrast colors with acid
dyes in the staining of animal tissues. Direct dyes are similar to
acid dyes and stain the proteins of connective tissues, muscles,
blood corpuscles, fungous mycelia, and parenchyma.

Sulfur dyes, which are direct dyes containing sulfur, are soluble
in alkaline solutions of sodium sulfide. They are especially suited
to the staining of blue-green algae. These pigments are insoluble
in glycerin, whereas the colors of most acid and basic dyes are ex-
tracted by this medium. Since the sulfur dyes are decolorized by
alcohol or acids they are not used on paraffin sections.
Mordant dyes, Colour Index Numbers.

781. Gallein (acid).

883. Gallocyanine (direct).
1049. Acid Alizarin Green G (acid).
1051. Alizarin Cyanine G (direct).
1239. Carmine (acid).
1246. Haematoxylin (acid).
The behavior of mordant dyes parallels the differences noted

between acid and direct dyes. The acid mordant dyes stain tissues

16 W. H. E M I G

taken from a fixative containing the salt of a heavy metal, mercuric
chloride, chromic acid, or potassium dichromate. The direct mor-
dant dyes have a more extensive application because they are not
adversely afTected by fixatives. Sections of paraffin materials are
left in 1 per cent ferric alum or 5 per cent ammonium alum for 30
minutes to an hour. After rinsing in distilled water, the section is
covered with the stain. Some of these dyes are inactive until they
are heated to 50° C. The depth of color in a finished mount may
be estimated from the section in water ; dehydration in alcohol ex-
tracts very little of the color. The time required in a mordant dye
differs: (1) Gallein one to ten minutes, (2) Gallocyanine one min-
ute to an hour, (3) Acid Alizarine Green G five minutes to an hour,
(4) Alizarine Cyanine G one minute to ten hours, (5) Carmine 30
minutes to 24 hours, (6) alum Haematoxylin 15 to 30 minutes, and
(7) iron Haematoxylin one to three minutes or sometimes as long
as 24 hours.

Vat dyes, as a class, are very fast to light. Their application is
limited, at present, to the staining of blue-green algae, page 64.

Basic dyes, Colour Index Numbers.

680. Methyl Violet.

740. Acridine Red.

752. Rhodamine 6G.

841. Safranine.

924. Methylene Green.

927. New Methylene Blue.

All of these dyes are soluble in water. The stain should con-
tain 0.5 per cent of the dye, except Methyl Violet where only 0.1
per cent is needed. Acetic acid and ammonium alum are the
chemical assistants.

The characteristic properties of basic dyes are derived from the
radicals -NH 2 , -N(CH 3 ) 2 , or -N(C 2 H )=- They are usually
marketed in the form of hydrochloride salts. In general, they
stain plant tissues 25 times more rapidly than the acid dyes. The
rate at which the staining process takes place depends on the extent
of ionization of the dye molecule. In the presence of a small
quantity of alkalies the color reaction is intensified, as in the stain-
ing of bacteria with alkaline Methylene Blue. The presence of

STAIN TECHNIQUE 17

an acid retards the speed of staining, ammonium alum produces a
color more fast to washing, and the two together in the stain pro-
vide a better differentiation of minute cell structures. Basic dyes
have an affinity for tissues containing tannins, resins or fatty acids ;
for spores, lignified xylem, cellulose, fibers, and cork. The chroma-
tin and chromosomes are stained before the cytoplasm is affected.

An unfamiliar basic dye is used for one minute. If the color is
too light, try one hour and then 24 hours. Whenever the color is
too dark at the end of one minute, leave the material in the stain
for five seconds. With two or three tests it is possible to approxi-
mate the time interval that gives the desired result. A clear color
is obtained with Rhodamine 6G in 12 to 24 hours, Safranine in
15 minutes to 24 hours, Methylene Green 15 to 30 minutes, New
Methylene Blue in five to ten minutes, and Methyl Violet in three
seconds to ten minutes.

Nomenclature of dyes. Under the above system of classify-
ing the commercial dyes, a very convenient mode of indicating the
particular shade of dyestuff by a letter or number denotes: A,
extra concentrated, as in Safranine A ; B, blue, as in Niagara Sky
Blue 6B; D, direct, in Alkaline Green D; G, (gelb) yellow, as in
Pyronine 2G ; O, concentrated, in Safranine O ; R, red, an Acridine
Yellow R; S, (sauer) acid, as in Fuchsine S; X, concentrated, in
Safranine X; Y, yellow, as in Bismarck Brown Y; and the nu-
merals, I, II, III, in Orange I, Orange II, Orange III, and Orange
IV which indicate a series of shades of one color.

Chapter IV
STAINING WITH CONTRAST DYES

The correlation between tissue elements and particular classes
of dyes serves as a working basis for the selection of combinations
of contrast dyes. The composition of organic materials may be
regarded as comprehensive groups of carbohydrates, carbohy-
drates and proteins, or only proteins. Carbohydrates are stained
more readily with basic dyes but proteins are differentiated more
easily with various types of acid dyes. This initial relation be-
tween tissues and stains implies : ( 1 ) acid dyes for embryonic or
mature animal tissues and meristematic parenchyma, (2) basic and
acid dyes for stem, root, or leaf, and (3) basic dyes for sections of
wood. As a general rule the greater the inherent difference in
cellular structure the greater should be the chemical differences of
the dyes present in the combination.

The selection of any combination of two or more dyes de-
pends not so much on whether the section is animal or plant tissue
as on whether the section consists of similar or unlike substances.
In most sections of animal tissues, fungi, and meristems, the varia-
tions are usually between types of proteins. These similar sub-
stances provide different colors more easily in mixtures of closely
related acid dyes. Although the color of nuclei may be intensified
with basic stains, clear contrasts of protein elements are better in
acid dyes with basic properties, that is, direct dyes which possess
an amino radical. Whenever cellulose assumes a dominant part of
vascular plants a basic dye is necessary. Then, the red basic
Rhodamine 6G, Safranine, Acridine Red, or Basic Fuchsine may
be used with a counterstain such as Aniline Blue, Niagara Sky
Blue, Fast Green, or Haematoxylin.

Many stains either lack a suitable counterstain or they dif-
ferentiate with few other dyes. Sun Yellow, a direct dye, gives
a clear contrast with Niagara Sky Blue or New Methylene Blue,
but blends on plant tissues into a dull gray-violet with Methyl
Violet. Biebrich Scarlet, an acid dye, differentiates sharply with
either of the blue dyes or the violet basic dye.

18

STAIN TECHNIQUE 19

Selecting a combination of stains. Test the object with a
stain from each of the four classes : acid, direct, mordant, and
basic. If the best colors are given with basic and mordant stains,
the combination of Safranine and Haematoxylin may be tried.
Should the direct dye work better than Haematoxylin, use
Safranine and Niagara Sky Blue. Many sections of growing tis-
sues fail to differentiate with a combination containing a basic dye.
If both the acid and direct stains provide clear colors on the test
material, the desired contrast is obtained by using either an acid
and a direct dye or two acid dyes of contrasting colors. The direct
dyes may be used as contrast stains with either an acid or a basic
dye.

Combinations of two stains.

Rule : Use the lighter stain first.
Example No. 1 — Crocein Scarlet MOO, C. I. No. 252, and Niagara

Sky Blue, C. I. No. 520.

This red acid dye and blue direct dye provide an adequate con-
trast on small invertebrates, paraffin sections of animal tissues,
meristems, or on fungi. The combination of stains may be used in
two ways, either separately or mixed in solution.
I. Two dyes used separately.

1. Ten minutes in Crocein Scarlet MOO.

2. Rinse the material in distilled water.

3. One minute in Niagara Sky Blue.

4. Rinse the material in distilled water.

5. Objects used as whole mounts are given three minutes
in each grade of alcohol: 35, 50, 70, 85, 95, and abso-
lute alcohol. Paraffin sections are transferred from
water to 95 per cent alcohol and then into absolute
alcohol, one minute in each.

6. Materials used as whole mounts need three minutes
in each mixture: \ xylene, \ xylene, J xylene and
xylene. Paraffin sections are given one, two, three,
and five minutes respectively in the alcohol-xylene
mixtures.

7. Whole mounts are infiltrated gradually in balsam.

20 W. H. E M I G

Cleared sections are covered with a drop of balsam
and a dry cover glass is added.
II. Two dye-solutions mixed.

1 . Five minutes in a mixture of ten parts Crocein Scarlet
MOO and one part Niagara Sky Blue.

2. Rinse the material in distilled water.

3. Follow the instructions given in the previous outline
schedule beginning with dehydration.

Example No. 2 — Sun Yellow, C. I. No. 620, and New Methylene

Blue, C. I. No. 927.
This combination of a direct yellow and a basic blue dye is
especially useful on plant tissues; it may be tried on invertebrate
animals.

1 . The material is given 24 hours in Sun Yellow.

2. Rinse the objects in distilled water.

3. Counterstain in New Methylene Blue, five minutes.

4. Rinse the material in distilled water.

5. Follow the usual laboratory procedure beginning with
dehydration.

The two dyes cannot be mixed because a precipitate forms
as soon as the stains are combined. The length of time the tissue
remains in New Methylene Blue modifies the results giving either
yellow and green, green and blue, or the three colors, yellow, green,
and blue.

Example No. 3 — Rhodamine 6G, C. I. No. 752, and Aniline Blue,

C. I. No. 707.

In staining mature vascular plants, the tissues are left for 24
hours in Rhodamine 6G, a bright red basic dye that does not over-
stain. The principal problem of technique at this stage is the
addition of enough Aniline Blue so that the red and blue are of
equal density. Use Aniline Blue on one stem section for a minute,
on another for two minutes, and on a third for five minutes. These
three sections in xylene serve as a comparison in determining the
correct time interval to be used.

If a counterstain covers all of the first color within a few sec-
onds, the counterstain is too intense. Under this circumstance, a

STAIN TECHNIQUE 21

weaker counterstain should be tried or the first stain should be
changed to a more intense dye of the same color. Blue dyes that
do not act so rapidly as Aniline Blue are Niagara Sky Blue and
alum Haematoxylin. If Rhodamine 6G does not give enough
color, the more intense Saf ranine or Acridine Red should be tried.
Combinations of three stains. The technique of applying
three of four dyes is nearly the same as that employed with com-
binations of two stains. Only two solutions are needed when two
or more dyes from the same group, either acid or basic, are mixed
and used as one stain. The proportionate amount of each dye in
a mixed stain is calculated either by the spot test or the time test.

1. Spot test using basic dyes. On filter paper note the contrast
between the red from a drop of Safranine and the violet from a
drop of Methyl Violet. For the first test try equal quantities of
Safranine and Methyl Violet ; compare a drop of this mixed stain
with the spots of the separate dyes on the filter paper. If the spot
given by a drop of mixed dyes shows a color intermediate between
the colors of the two original stains, the mixture contains the
proper amount of each. Should one color predominate in the test
on paper, the proportions are changed until the result is a blend of
the two original colors. As a rule, three parts of Safranine will
neutralize one part of Methyl Violet.

Staining procedure for mature plant tissues. Stain the mate-
rial for 24 hours in Orange II, rinse in distilled water, and follow
with the mixture of three parts Safranine and one part Methyl
Violet. Again rinse the section in distilled water, transfer it to
95 per cent alcohol, complete the dehydration in absolute alcohol,
and clear in xylene. During the counterstaining, a longer interval
in the mixed stain yields more violet while less time increases the
red.

2. Time test using acid dyes. Note the different time intervals
needed for staining a section of an earthworm in Sun Yellow,
Orange II, and other acid dyes.

Sun Yellow, C. I. No. 620, 10 minutes.
Orange II, C. I. No. 151, 5 minutes.
Crocein Scarlet MOO, C. I. No. 252, 5 minutes.
Biebrich Scarlet, C. I. No. 280, 2 minutes.

22

W. H. E M I G

Crocein Scarlet 7B, C. I. No. 286, 2 minutes.

Acid Fuchsine, C. I. No. 692, 30 seconds to 1 minute.
Whenever two of these dyes are mixed the amounts needed
are in direct proportion to the time interval required for the stain-
ing of each alone. Thus, mix ten parts of Sun Yellow with five
parts of Orange II, ten parts of Sun Yellow with one part of Acid
Fuchsine, or five parts of Orange II with one part of Acid
Fuchsine. In the following outline any of the eight mixed stains
may be tried with one of the three counterstains.

Counter stains

( 1 ) Aniline Blue

(2) Niagara Sky Blue

(3) New Methylene Blue

Mixed stains

Sun Yellow (10) and
Orange II (5)

Sun Yellow (10) and
Crocein Scarlet MOO (5)

Sun Yellow (10) and
Biebrich Scarlet (2)

Sun Yellow (10) and
Crocein Scarlet 7B (2)

Sun Yellow (10) and
Acid Fuchsine ( 1 )

Orange II (5) and
Biebrich Scarlet (2)

Orange II (5) and
Crocein Scarlet 7B (2)

Orange II (5 to 10) and
Acid Fuchsine ( 1 )

Staining procedure. Using sections of an earthworm, try one
of the mixed stains such as Sun Yellow and Orange II for five to
ten minutes. If the first mixed stain does not produce two colors
on the section, try another mixture. When two colors are ob-
tained, the mixture may be used with a counterstain. The problem
at this point is to find which of the three blue counterstains will
give the best result; it is taken for granted that from sufficient
experience in handling double stains the principle involved in the
procedure of counterstaining is understood.

STAIN TECHNIQUE 23

a) On animal tissues. Try the mixed stain for five minutes,
rinse the section in distilled water, and counterstain in Niagara
Sky Blue for one minute or in Aniline Blue for 30 seconds ; it may
be necessary to shorten or to increase the time in Niagara Sky Blue.
Any one of the eight mixed stains may be tried on sections of
earthworm and counterstained in either Niagara Sky Blue or in
Aniline Blue.

b) On meristematic plant tissues. Use the mixed stain for
one hour, rinse in distilled water, and counterstain in one of the
blue dyes for one minute.

c) On mature plant tissues. Use the mixed stain for 24 hours.
If there is an abundance of cellulose in the section, New Methylene
Blue for five minutes is the best counterstain. On fungous tissues
either Niagara Sky Blue or Aniline Blue gives a better contrast
than New Methylene Blue.

Combinations of four stains.

Mixed stains Counter stains

Sun Yellow ( 10) , ( 1 ) Aniline Blue

Orange II (5), and

Biebrich Scarlet (2) (2) Niagara Sky Blue

Sun Yellow (10) (3) New Methylene Blue

Orange 11 (5), and
Crocein Scarlet 7B (2)

Sun Yellow (10),
Orange II (5), and
Acid Fuchsine (1)

Staining procedure. On invertebrate animals use the first-men-
tioned mixed stain for five minutes and counterstain either in
Niagara Sky Blue for 30 seconds or in Aniline Blue for 15 seconds.
On sections of vertebrate tissues the second- or third-listed stain
may be tried for five minutes ; counterstain either in Niagara Sky
Blue or in Aniline Blue.

On sections of mature plant tissues use the first or third
mixture for 24 hours and counterstain in New Methylene Blue
for five minutes.

24 W. H. E M I G

A combination of four dyes should be used only on appropriate
materials consisting of a mass of different tissues. Whenever the
object presents a uniform structure one or two stains may be
sufficient. In searching for a better technique, keep in mind the
correlation between dyes and tissue elements. Note the differences
between acid, direct, mordant, or basic stains and determine the
adaptability of the material by testing separately each of the dyes
present in the combination.

Chapter V
BIOLOGICAL STAINS

A. Synthetic Organic Dyes, Classes I to VI

The synthetic coal-tar colors are classified on the basis of
molecular structure. The terminology of the functional parts of
the dye molecule is that proposed by Otto N. Witt in 1876. ( The
simplest compounds which illustrate the evolution of a dye are
derived from benzene. Particular radicals capable of imparting
color to aromatic compounds are called chromophores. The
chromophore - N0 2 added to a molecule of benzene yields nitro-
benzene, a chromogen. The color-bearing nitrobenzene does not
possess staining properties until it is transformed into a compound
capable of electrolytic dissociation. Salt-forming groups called
auxochromes, either hydroxyl or amino radicals, change an inert
chromogen to an acid or a basic dye respectively. Nitrophenol is
a simple acid dye containing the chromophore — N0 2 and the auxo-
chrome - OH ; nitroaniline is a simple basic dye including the
chromophore - N0 2 and the auxochrome - NH,.

The discussion of the separate dyes specifies the Colour Index
Number, the name most frequently used in American literature,
synonyms, fastness to light, the formula for the stain, and the
manner of application. The arrangement of the dyes follows the
classification of dyestuffs as given in the Colour Index, published
by the Society of Dyers and Colourists, Bradford, England, 1924.
Each coal-tar dye is given a separate number and is placed in one
of 26 classes according to the kind of chromophore in the dye
molecule ; the dye of the simplest structure is first in each class.

The comparative fastness of each dye is given on a scale of 1 to
5 for paraffin sections ten microns thick. A fastness of 1 indicates
a color that does not fade after 50 hours exposure to direct sun-
light at midday during the summer months. For the value 2, the
color fades slowly but lasts more than 50 hours on exposure to sun-
light. In group 3, the color disappears within 50 hours although
it is still evident at the end of 25 hours. Pigments that fade within

25

26 W. H. E M I G

25 hours are placed in group 4. Group 5 includes extremely
fugitive colors. Dyes with a light fastness of 4 or 5 have little
value for permanent slides.

Class I. Nitroso Colouring Matters

5. Naphthol Green B is an acid dye, fastness to light 2; J
gram in 100 ml. of distilled water and 1 ml. of 1 per cent sulfuric
acid. In commercial dyeing the nitroso dyes are applied using an
iron salt in the dye bath but in staining tissues a darker green is
obtained by using the iron salt separately as if it were a mordant.
A deficiency of tinctorial properties limits the use of Naphthol
Green B on paraffin sections except in plant tissues consisting of
dense spore masses.

Mordant the sections of an aethalium of Fuligo, a Myxomy-
cete, in 1 per cent ferric ammonium sulfate for 30 minutes, rinse in
distilled water, and stain for from one to 24 hours. On whole
mounts of fungous mycelia five to ten minutes is sufficient with
ferric alum as a mordant, 24 hours without the mordant.

Obelia, Sertularia, Hydra, and animals of similar size are placed
in 1 per cent ferric alum for 30 minutes. After a rinse in dis-
tilled water the objects are stained dark green in ten minutes ; thick
specimens are easily overstained. Without the iron alum mordant
six to ten hours is needed. The invertebrates stained in Naphthol
Green B ten minutes may be counterstained, after a rinse in dis-
tilled water, for 15 minutes in Direct Fast Scarlet 4BS, C. I. No.
327.

Class II. Nitro Colouring Matters

7. Picric Acid, trinitrophenol, is an acid dye, light 2 to 5; ■£
gram in 95 ml. of distilled water, 5 ml. of ^ per cent potassium
dichromate, and \ ml. of acetic acid. Although Picric Acid is the
oldest of the artificial dyestufTs it is rarely used commercially as a
dye except in small quantities in mixtures for shading. From an
aqueous stain animal and plant tissues are pale yellow. With the
aid of potassium dichromate and acetic acid the newly developed
pigment appears a fast yellow which, in most tissues, is too light.

9. Martius Yellow, Aniline Yellow, Naphthylamine Yellow is

STAIN TECHNIQUE 27

an acid dye, light 4 to 5. The absence of a bitter taste accounts
for the former use of this poisonous dye in coloring starchy food
such as vermicelli and macaroni. Pianese includes Martius Yellow
with Malachite Green and Acid Fuchsine for the differential stain-
ing of cancerous tumors. Other technicians use the triple stain
for fungous parasites on plants. There is no need for Martius
Yellow in the mixture when the yellow can be traced to either a
chemical in the fixative or a natural pigment. Instead of Martius
Yellow use Sun Yellow, C. I. No. 620.

10. Naphthol Yellow S, Acid Yellow is an acid dye, light 3 to
4. Only a light color is obtained unless acetic acid and potassium
dichromate are present in the stain. It is more fast than Martius
Yellow and more soluble in water ; acids cause no precipitates in its
solution. Its principal value is for the coloring of foods such as
noodles and macaroni.

Class III. Monoazo Colouring Matters

Although the azo chromophore is basic in character, the azo
dyes may be either basic or acid according to the auxochromes
present. The number of chromophores in the dye molecule
determines the classification as monoazo, disazo, trisazo, or polyazo
dyes. Those azo dyes which are insoluble in water or alcohol may
be used as fat soluble stains. Azo dyes differ through all degrees
of fastness to light ; many are fugitive whereas others can satisfy
the most exacting requirements in this respect. In some instances
the addition of potassium dichromate to the stain increases the
intensity of the color and its fastness.

16. Acid Yellow, Fast Yellow S is a direct dye, light 2 to 3;
i gram in 95 ml. of distilled water, 1 ml. of 1 per cent sulfuric acid,
and 5 ml. of \ per cent potassium dichromate ; filter. Sections of
animal or plant materials are stained a clear fast orange-brown
that is not very brilliant.

20. Chrysoidine Y, and 21. Chrysoidine R are basic dyes,
light 4; the former dye is more soluble. The stain decomposes
slowly on standing. These dyes are similar to Bismarck Brown in
color but they are not its equal in fastness. Sharp contrasts are

28 W. H. E M I G

obtained by cotmterstaining with direct blue dyes such as Niagara
Sky Blue.

27. Orange G, Acid Orange G is an acid dye, light 3 to 4; -J
gram in 100 ml. of distilled water and 1 ml. of 1 per cent sulfuric
acid. The fastness of Orange G to washing is greatly increased
by the presence of acids. If the material is treated with -J per
cent potassium dichromate, the stain reacts more rapidly; potas-
sium dichromate cannot be added to the dye. Best results occur
on tissues fixed in a solution containing picric acid; with other
types of fixatives Orange II is better.

Orange G is used extensively on animal tissues as a counter-
stain in conjunction with iron Haematoxylin. Because the iron
alum present in the tissues as a mordant for Haematoxylin also
serves as a chemical assistant for Orange G, the stain need not
contain sulfuric acid. Since the orange tends to obscure a portion
of the finer details of cell structure, many technicians prefer iron
Haematoxylin without a counterstain.

Mallory's connective tissue stain includes Orange G, Aniline
Blue, and Acid Fuchsine. On many sections, Aniline Blue fails to
differentiate well with Orange G; the colors may be striking but
they usually lack clear definition. The deficiencies inherent in
Mallory's triple stain are also found in sections stained with Ehr-
lich's Orange G, Methyl Green, and Acid Fuchsine.

A more satisfactory combination of stains is a mixture of nine
parts Orange G and one part Acid Fuchsine for two minutes which
is followed by Aniline Blue for 15 seconds on most sections of
animal tissues. As an alternative procedure try Orange II and
Acid Fuchsine followed by Niagara Sky Blue.

Flemming's triple stain is frequently used on all kinds of plant
tissues. Saf ranine is applied first ; the excess of red is removed in
acid-alcohol. A brief stain in Gentian Violet is followed by dehy-
dration and then Orange G in clove oil is added. These three dyes
may be applied in a simpler manner. Stain the plant tissue in
Orange G, or preferably Orange II, and then apply the counter-
stains in a mixture of three parts Saf ranine and one part Methyl
Violet, page 21.

STAIN TECHNIQUE 29

In the contrast staining of plant tissues mordant the material
for 15 minutes in J per cent potassium dichromate, rinse in dis-
tilled water, use Orange G overnight, rinse again with distilled
water, and follow with : ( 1 ) one minute in Methyl Violet, C. I. No.
680, (2) five minutes in Niagara Sky Blue, C. I. No. 520, or (3)
five minutes in New Methylene Blue, C. I. No. 927.

28. Ponceau G, 2G, Scarlet 2G is an acid dye, light 3 to 4 ; J
gram in 100 ml. of distilled water and 1 ml. of 1 per cent sulfuric
acid. Many tissues are stained fuchsia. Materials previously
treated with potassium dichromate are scarlet and more fast to
washing. One of the "poppy" dyes, it is similar to Ponceau 3R, C. I.
No. 80, and Crocein Scarlet MOO, C. I. No. 252. On animal tissues
use Ponceau G in the same manner as described in the technique for
Crocein Scarlet MOO.

On plant tissues use the stain for 12 to 24 hours and follow
with: (1) Victoria Blue B, C. I. No. 729, five minutes for reddish
brown and blue, (2) Methyl Violet, C. I. No. 680, one minute, or
(3) Niagara Sky Blue, C. I. No. 520, five minutes for reddish
brown and blue.

29. Chromotrope 2R is an acid dye, light 4. There are many
acid dyes of a nondescript fuchsia; this one is very fugitive to
light. As successive azo dyes are considered notice the progressive
increase of color intensity.

30. Azofuchsine, Acid Azofuchsine is an acid dye, light 3.
The bright fuchsia obtained on plant tissues is changed to bordeaux
with either a mordant or an after-treatment of potassium
dichromate.

31. Pontacyl Carmine 2G (Du Pont) is an acid dye, light 3.
This fuchsia pigment is more intense than the color obtained with
Azofuchsine. After a potassium dichromate mordant, the color
changes to bordeaux. Fungous mycelia treated with a mordant
remain light fuchsia.

36. Alizarin Yellow G, and 40. Alizarin Yellow R are mor-
dant dyes, light 5. The color on paraffin sections is fugitive. The
alcoholic solution of the dye stains blue-green algae a brilliant yel-

30 W. H. E M I G

low-brown that gradually disappears when the algae are trans-
ferred to glycerin.

44. Para Red, Azophor Red PN is a developed dye which is
not adaptable to paraffin sections. It may be produced on whole
mounts by first using an alkaline beta-naphthol solution and then
following with an ice cold diazotized paranitroaniline. The
orange-red pigment lacks sufficient density for whole mounts. Be-
cause Azophor Red is insoluble in water or alcohol, it cannot be
used as a stain.

57. Pontacyl Carmine 6B (Du Pont) is an acid dye, light 3.
Thin sections of tissue are fuchsia with a decided bluish cast. A
potassium dichromate mordant darkens the color considerably.
Numerous tints and shades which are valued in commercial dyeing
are not so important in microtechnique where the bright primary
colors are preferred.

73. Sudan II may be used as a fat stain; 1/10 gram in 50 ml.
of 95 per cent alcohol and 50 ml. of 1 per cent sulfuric acid.
Freehand sections of fresh tissue containing fat become orange-
red at 50° C. although starches and proteins remain unchanged.

74. Scarlet R is an acid dye, insoluble in water. Because it is
soluble only in alkaline 50 per cent alcohol, the dye is unsuited for
staining paraffin sections.

79. Ponceau R, 2R, Scarlet R, 2R is an acid dye, light 4.
Plant tissues are stained a bright red that contrast well with green
basic dyes. It is similar to Ponceau 3R which is more fast. Ani-
mal tissues are stained in the same manner as described in the
technique for Ponceau 3R, C. I. No. 80.

80. Ponceau 3R is an acid dye, light 3 ; J gram in 100 ml. of
distilled water and 1 ml. of 1 per cent sulfuric acid. On sections
of animal tissues use a mixture of three parts Sun Yellow, C. I. No.
620, and one part Ponceau 3R for five minutes ; follow with ( 1 )
Niagara Sky Blue, C. I. No. 520, for two minutes or (2) with
Aniline Blue, C. I. No. 707, one minute. The results are not
satisfactory on a tissue that has been fixed in either picric acid or a
chromium salt.

STAIN TECHNIQUE 31

On plant tissues the scarlet is similar to that of Biebrich Scar-
let. Leave the material in Ponceau 3R for 12 hours and counter-
stain with one of the following : ( 1 ) five minutes in Victoria Blue
B, C. I. No. 729, (2) five minutes in Brilliant Cresyl Blue, C. I.
No. 877, for reddish brown and violet, or (3) one minute in Methyl
Violet, C. I. No. 680, for fuchsia and violet.

88. Bordeaux, Azo Bordeaux, Acid Bordeaux is an acid dye,
light 3 ; \ gram in 100 ml. of distilled water and 1 ml. of 1 per cent
sulfuric acid. The fuchsia on plant materials blends with blue
dyes to give colors of little contrast; a fair differentiation is
obtained with Victoria Blue B, C. I. No. 729. Bordeaux is also
used for coloring foods.

101. Pontachrome Brown MW (Du Pont) is a mordant dye,
light 4. A clear brown of moderate fastness is obtained on blue-
green algae with the alkaline solution of the dye. Neutral solu-
tions in alcohol give a reddish brown on paraffin sections.

120. Coccinine is an acid dye, light 3 to 4. Materials in
paraffin sections are stained a pale pink which is not the equal of
Biebrich Scarlet or of the Crocein Scarlets.

133. Janus Green B is a basic dye, light 4. This dye, now
obsolete, was formerly used on fabrics. With staining properties
decidedly different from most basic dyes, it can be applied on cotton
in an acid bath. In whole mounts of algae the stain is poly-
chromatic; in a stem section all of the cells are either blue or
blue-green. If it were more fast Janus Green B would be a good
stain to use on freehand sections.

138. Metanil Yellow is an acid dye, light 4 to 5. Cells of
parenchyma are stained a fugitive yellow. Upon the addition of
potassium dichromate the pigment becomes light brown.

142. Methyl Orange, Helianthine, Orange III is an acid dye,
light 4. Because Methyl Orange is not affected by carbonates or
weak organic acids it is used principally as an indicator. On
paraffin sections it is similar to Orange G in both color and fast-
ness.

143. Orange IV, Tropaeolin OO is an acid dye, light 4 to 5.
Cells of parenchyma are yellow in acid solutions of the dye but

32 W. H. E M I G

light brown in the presence of potassium dichromate. Without
the addition of acids the dye has slight staining properties.

148. Tropaeolin O is an acid dye, light 3 ; J gram in 95 ml. of
distilled water, 1 ml. of 1 per cent sulfuric acid, and 5 ml. of ^ per
cent potassium dichromate. Vascular plant tissues are stained yel-
lowish orange to brown of moderate fastness. Blue-green algae
are clear yellow in alkaline solutions of the dye.

151. Orange II, Acid Orange II, Gold Orange, Tropaeolin
OOO No. 2 is an acid dye, light 3 to 4 ; ^ gram in 100 ml. of dis-
tilled water and 1 ml. of 1 per cent sulfuric acid.

In staining vertebrate tissues or whole mounts of invertebrates
use a mixture consisting of two parts Sun Yellow, C. I. No. 620,
and one part Orange II for five to ten minutes and counterstain
either in Aniline Blue, C. I. No. 707, for 15 seconds or in Niagara
Sky Blue, C. I. No. 520, for 30 seconds. In a combination with
Acid Fuchsine, C. I. No. 692, use nine parts of Orange II and one
part Acid Fuchsine for two minutes and follow with Niagara Sky
Blue for 30 seconds. If the tissue differentiates clearly, the red,
orange, and blue provide a striking contrast.

On sections of animal or fungous tissues use a mixture of Sun
Yellow and Orange II for 10 minutes and counterstain in Niagara
Sky Blue. Aniline Blue may be tried as a counterstain either with
Orange II alone or with the mixture of Sun Yellow and Orange
II. A very good triple stain on plant tissues includes a mixture of
two parts Orange II and one part Biebrich Scarlet for 24 hours.
After a rinse in distilled water, counterstain meristems with
Niagara Sky Blue; woody tissues may be counterstained in
Niagara Sky Blue, Methyl Violet, C. I. No. 680, or New Methylene
Blue, C. I. No. 927.

Treat mature plant tissues with \ per cent potassium dichro-
mate for 15 minutes, rinse in distilled water, and stain for 24 hours
in Orange II. The counterstains are: (1) Aldehyde Green, C. I.
No. 676a, five to ten minutes for blue-green and orange, (2)
Thionine, C. I. No. 920, for blue and orange, (3) Victoria Blue B,
C. I. No. 729, five minutes, (4) Methyl Violet one minute, or (5)
New Methylene Blue one to five minutes.

STAIN TECHNIQUE

33

Combinations of Stains

Double stains
Orange II .

X

Triple stains

Orange II (5) and
Biebrich Scarlet (2)

X

Orange II (10) and

Acid Fuchsine ( 1 ) X

Quadruple stains
" Sun Yellow (10),
Orange II (5), and
Crocein Scarlet MOO (5) or
Biebrich Scarlet (2) or
Acid Fuchsine ( 1 ) ... X

Counter stains

(1) Aldehyde Green

(2) Aniline Blue

(3) Methyl Violet

(4) New Methylene Blue

(5) Niagara Sky Blue

(6) Thionine

(7) Victoria Blue B

(1) Methyl Violet

(2) New Methylene Blue

(3) Niagara Sky Blue

(4) Victoria Blue B

(1) New Methylene Blue

(2) Niagara Sky Blue

(1) New Methylene Blue

(2) Niagara Sky Blue

(3) Aniline Blue

161. Orange R is an acid dye, light 3 to 4; J gram in 100 ml.
of distilled water and 1 ml. of 1 per cent sulfuric acid. Stain sec-
tions of animal tissues in a mixture of two parts Sun Yellow, C. I.
No. 620, and one part Orange R for ten minutes, rinse in distilled
water, and counterstain in Erie Violet BW, C. I. No. 387, eight
minutes for orange, red, and violet.

On sections of animal or fungous tissues use a mixture of Sun
Yellow and Orange R for 10 minutes ; counterstain in Niagara Sky
Blue, C. I. No. 520, for one or two minutes. This combination
stain does not give satisfactory colors on tissues taken from a fixa-
tive containing picric acid. On plant tissues, Orange R has a more
reddish shade than Orange II; the counterstains are: (1) Methyl
Violet, C. I. No. 680, (2) Aldehyde Green, C. I. No. 676a, (3)

34 W. H. E M I G

Thionine, C. I. No. 920, (4) New Methylene Blue, C. I. No. 927,
or (5) Victoria Blue B, C. I. No. 729.

168. Superchrome Garnet Y (NAC) is a mordant dye, light

3. Paraffin sections of plant tissues are stained orange-yellow to
brown. The color becomes darker with an after-treatment in
potassium dichromate. Blue-green algae are stained orange-red
in an alkaline solution of the dye at 50° C. The pigment gradually
disappears in glycerin.

169. Superchrome Violet B (NAC) is an acid mordant dye,
light 3. Paraffin sections of plant tissues appear bright pink ; they
are more fast to washing with an after-treatment in 5 per cent
ammonium alum. A mordant of potassium dichromate and acid
potassium tartrate gives a reddish brown on parenchyma. Blue-
green algae in an alkaline bath containing potassium dichromate,
heated to 50° C, are dark carmine. The color disappears slowly
in 10 per cent glycerin.

170. Superchrome Black PV (NAC) is an acid mordant dye.
Paraffin sections of plant tissues stain a dull bordeaux ; with a mor-
dant, the color is bordeaux to brown. Blue-green algae in a hot
alkaline bath are violet to blue-black; the pigment is not fast in
glycerin.

175. Naphthylamine Brown is an acid dye, light 3 to 4.
Plant tissues stain light brown to rich red-brown ; in the presence
of potassium dichromate they are darker brown.

176. Fast Red A is an acid dye, light 4. This stain does not
differentiate well but blends with blue dyes.

179. Azorubine is an acid dye, light 4. Commercially, Azoru-
bine is used as a substitute for the more expensive Acid Fuchsine.
Plant tissues are stained brilliant red that contrasts well with blue
dyes. Unfortunately, the color fades rapidly.

184. Amaranth, Naphthylamine Red is an acid dye, light 3 to

4. This dye is used principally as a food color.

185. Brilliant Scarlet 3R is an acid dye. Most materials are
stained pink which is too pale to be useful. The stain becomes
moldy on standing.

STAIN TECHNIQUE 35

207. Lanacyl Violet B is an acid dye, light 3 ; i gram in 95
ml. of distilled water, 1 ml. of 1 per cent sulfuric acid, and 5 ml. of
J per cent potassium dichromate. Parenchymatous tissue, cell
walls, and fungous mycelia are stained clear blue-violet very
similar in appearance to tissues taken from alum Haematoxylin.

210. Lanacyl Blue BB is an acid dye. Cell walls and paren-
chymatous cells are stained light blue. The dye is not so good as
other blue acid dyes.

211. Methyl Red is used principally as an indicator. It has
little value as a stain.

Class IV. Disaso Colouring Matters

248. Sudan III is unsuited for staining paraffin sections.
Blue-green algae are stained a fugitive orange-red in an alkaline
solution of the dye. As a fat stain this dye is used in the same
manner as Sudan IV, C. I. No. 258.

252. Crocein Scarlet MOO, Carmoisine is an acid dye, light
3; J gram in 100 ml. of distilled water and 1 ml. of 1 per cent sul-
furic acid. On animal tissues this stain is more valuable than
Acid Fuchsine. Use Crocein Scarlet MOO for 10 minutes and
counterstain in Niagara Sky Blue, C. I. No. 520, for one minute.
With a mixture containing two parts Sun Yellow, C. I. No. 620,
and one part Crocein Scarlet MOO for 10 minutes, the counter-
stains are : ( 1 ) Niagara Sky Blue one minute for red, green, and
blue, (2) Aniline Blue, C. I. No. 707, which is not so selective as
Niagara Sky Blue, 15 seconds, or (3) Erie Violet BW, C. I. No.
387, five minutes for red, violet, and green.

Sections of fungous tissues remain in Crocein Scarlet MOO
for 10 minutes and are counterstained in Niagara Sky Blue one
minute. Any of the combinations suggested for animal tissues
should be tried on fungi. On vascular plants this bright red dye
differentiates well with blue basic stains. It is similar to Azoru-
bine in color and staining reactions but is more fast. It counter-
stains well with (1) Aldehyde Green, C. I. No. 676a, one to ten
minutes, or (2) Opal Blue, C. I. No. 689, 30 minutes with a direct
transfer from the stain to 95 per cent alcohol. Fairly good con-

■^

36 W. H. E M I G

trasts are obtained with Thionine, C. I. No. 920, Methylene Green,
C. I. No. 924, or New Methylene Blue, C. I. No. 927.

258. Sudan IV is insoluble in water but soluble in alcohol, red-
dish violet in alkaline solutions; 1/10 gram in 50 ml. of 95 per cent
alcohol and 50 ml. of 1 per cent sulfuric acid ; heat to 50° C. From
paraffin sections of various tissues the fats and oils are extracted
with alcohol and xylene. Those structures which formerly con-
tained fats are readily stained with either basic or direct dyes.

Freehand sections of fresh tissue are placed in Sudan IV at 50°
C. for an hour or longer. The tissues containing fats become
bright red which is slightly more intense and darker than the color
produced by Sudan II, C. I. No. 73, or Sudan III, C. I. No. 248.
Tissues consisting of carbohydrates or proteins remain unchanged.

266. Janus Red B is a basic dye, light 4. Parenchyma be-
comes fugitive pink to red depending on the length of time in the
stain.

280. Biebrich Scarlet, Crocein Scarlet 5 R is an acid dye, light
3; ^ gram in 100 ml. of distilled water and 1 ml. of 1 per cent sul-
furic acid. This is the best red acid dye for staining plant tissues.
It is more scarlet and more fast than Azorubine; it may be used in
techniques calling for Acid Fuchsine. Tissues fixed in formalin,
picric- formalin-acetic, or in Zenker's solution take a brilliant color ;
after Gilson's fixative the stain is not always satisfactory. As a
rule Biebrich Scarlet does not overstain mature tissues but differen-
tiates easily with green, blue, or violet basic dyes.

Use Biebrich Scarlet on vascular tissues 24 hours and counter-
stain with: (1) Methyl Violet, C. I. No. 680, one to three minutes,
(2) Victoria Blue B, C. I. No. 729, five minutes, (3) New
Methylene Blue, C. I. No. 927, five minutes, or (4) Aldehyde
Green, C. I. No. 676a, five minutes for a mixture of red, green, and
greenish black.

Except for certain invertebrates Biebrich Scarlet is not so good
on animal tissues. On whole mounts of small animals use a mix-
ture of ten parts Sun Yellow, C. I. No. 620, and two parts Biebrich
Scarlet for five minutes ; follow with Niagara Sky Blue, C. I. No.
520, for 15 to 30 seconds.

STAIN TECHNIQUE

37

Combinations of Stains

Double stains
Biebrich Scarlet

X

Triple stains

Biebrich Scarlet (2) and
Orange II (5) X

Biebrich Scarlet (2) and
Sun Yellow (10) X

Quadruple stains

Biebrich Scarlet (2),

Orange II (5), and

Sun Yellow (10) X

Counter stains

(1) Aldehyde Green

(2) Aniline Blue

(3) Methyl Violet

(4) New Methylene Blue

(5) Niagara Sky Blue

(6) Victoria Blue B

(1) Methyl Violet

(2) New Methylene Blue

(3) Niagara Sky Blue

(1) New Methylene Blue

(2) Niagara Sky Blue

(1) New Methylene Blue

(2) Niagara Sky Blue

286. Crocein Scarlet 7 B is an acid dye, light 3 ; J gram in 100
ml. of distilled water and 1 ml. of 1 per cent sulfuric acid. This
stain has properties similar to those of Biebrich Scarlet except that
it is more fuchsia. It may be used on animal tissues instead of
Acid Fuchsine. On sections of animal tissues use one of the triple
stains. Place the material in a mixture of five parts Sun Yellow,
C. I. No. 620, and one part Crocein Scarlet 7B for five minutes;
the counterstains are: (1) Niagara Sky Blue, C. I. No. 520, one
minute, (2) Aniline Blue, C. I. No. 707, 30 seconds, (3) Direct
Green B, C. I. No. 593, two minutes, or (4) Erie Violet BW, C. I.
No. 387, eight minutes.

On mature plant tissues use Crocein Scarlet 7B for 24 hours
and counterstain with (1) Methyl Violet, C. I. No. 680, three min-
utes, (2) Victoria Blue B, C. I. No. 729, ten minutes, (3) Niagara
Sky Blue five minutes, or (4) Aniline Blue five minutes.

289. Tolyl Blue 5R is a mordant dye, light 3 ; J gram in 100
ml. of 1 per cent ammonium acetate. With potassium dichromate
as the mordant, tracheids, pollen, and fungous mycelia are blue in

38 W. H. E M I G

the stain containing acetic acid. These results indicate that the
dye is relatively inactive at room temperature. If the material is
mordanted in ferric ammonium sulfate and stained for three hours
at 50° C.j meristems are clear blue; brown and red algae are dark
blue. Nuclei become darker than the cytoplasm. In alkaline solu-
tions of the dye, blue-green algae stain a clear brilliant blue but the
color disappears in glycerin.

Sections of animal tissues are placed in the mordant 1 per cent
ferric alum for 15 minutes. After a rinse in distilled water, Tolyl
Blue 5R stains the material at 50° C. within five to ten minutes.
The sections are clear bright blue.

315. Wool Black B is an acid dye, light 3 to 4. Parenchyma
stains a clear blue; sclerenchyma is tinted green to dull greenish
black. In the presence of potassium dichromate, nuclei remain
uncolored but the cytoplasm stains blue-green.

326. Direct Fast Orange, Erie Fast Orange (NAC), Erie
Fast Scarlet YA (NAC) is a direct dye, light 3.

327. Direct Fast Scarlet 4BS, Pontamine Fast Scarlet 4BS
(Du Pont) is a direct dye, light 3; J gram in 95 ml. of distilled
water, 5 ml. of 5 per cent sodium sulfate, and \ ml. of acetic acid.
Direct Fast Orange and Direct Fast Scarlet differ only in color.
The two dyes are not so good on tissues taken from Gilson's or
Bouin's fixative. Whenever alcohol, formalin, and acetic acid are
used in one of the many modifications of fixatives, animal tissues
become brilliant orange-red in 15 to 30 minutes, but mature plant
tissues need an overnight stain for a full development of the color.
Fortunately, the dye does not overstain. It is fast to washing in
water, alcohol, ferric alum, and remarkably fast to acids, including
nitric acid.

Direct Fast Scarlet 4BS may be used as a general stain instead
of Carmine on paraffin sections of animal or fungous tissues. The
dye is most useful as a counterstain. The material is first stained
in iron Haematoxylin, differentiated in iron alum, and then
counterstained in Direct Fast Scarlet 4BS — animal tissues 15 to 30
minutes, meristematic plant tissues 30 minutes, mature plant
tissues overnight. The differential staining of parasitic fungi and

STAIN TECHNIQUE 39

plant host should be tried with alum Haematoxylin and Direct Fast
Scarlet 4BS. This latter combination of stains provides the con-
trast which has been attempted so often with Haematoxylin and
Congo Red. Should Direct Fast Scarlet 4BS be the first stain
used on mature plant tissues counterstain the material with Methyl
Violet, C. I. No. 680.

331. Bismarck Brown G, Y, and 332. Bismarck Brown R,
Vesuvine R are basic dyes, light 3; J gram in 95 ml. of distilled
water, 5 ml. of 5 per cent ammonium alum, and \ ml. of acetic acid.
An overnight stain on plant tissues gives an intense brown ; the R
shade is the darker. On paraffin sections, Bismarck Brown is not
equal to Acridine Orange in fastness.

353. Pontamine Fast Pink BL (Du Pont) is a direct dye,
light 3 to 4. A fuchsia pigment is obtained on paraffin sections of
various plant tissues with an acid solution of the dye. With a
potassium dichromate mordant, parenchyma is stained brick red.
In alkaline solutions of the dye, blue-green algae are colored a fugi-
tive pink.

364. Brilliant Yellow is an acid dye, light 3. For most sec-
tions of materials embedded in paraffin the color is too light ; resin-
ous tissues stain bright yellow from an acid solution. Blue-green
algae are a clear yellow in alkaline solutions.

365. Chrysophenine is a direct dye, light 4 to 5. This dye is
used as a substantive yellow on fabrics for bronze shades. On
paraffin sections the stain is too light and too fugitive.

370. Congo Red is a direct dye, light 4 ; ^ gram in 95 ml. of
distilled water and 5 ml. of 5 per cent sodium sulfate. On sections
of animal tissues Congo Red is used as a contrast stain with
Haematoxylin. The color is not uniformly distributed on most
objects because the stain does not have a specific selection for any
particular tissue.

If Congo Red were more fast to light it would be very useful
for staining fungous mycelia within a plant host. With materials
fixed in Gilson's solution the host appears purple in alum Haema-
toxylin; after the section is rinsed in distilled water, the fungous
parasite is stained in Congo Red for 24 hours. The dye is so

40 W. H. E M I G

soluble in water that the sections are dehydrated as quickly as pos-
sible after a direct transfer from the stain to 95 per cent alcohol.
The brick red of stained fungi changes to dull brown if sections are
left too long in xylene before mounting. Acids precipitate this
dye from solution and change the color from red to blue ; for that
reason Congo Red is sometimes used as an indicator. Instead of
Congo Red use Direct Fast Scarlet 4BS on either animal or plant
tissues.

375. Congo Corinth G, Erie Garnet B is a direct dye, light 4.
This dye is more orange and more fugitive than Congo Red.

387. Erie Violet BW (NAC) is an acid dye, light 2 to 3; i
gram in 100 ml. of distilled water and 1 ml. of 1 per cent sulfuric
acid. In sections of animal tissues Erie Violet BW may be used
as a counterstain with either Crocein Scarlet MOO, C. I. No. 252,
or Crocein Scarlet 7B, C. I. No. 286.

On plant tissues the cytoplasm of meristematic cells is stained
violet while the nuclei remain uncolored. Storage parenchyma
and cells containing resin are colored reddish violet to violet-black.

394. Erie Violet 3R (NAC) is a direct dye, light 3. This dye
is not so good as Erie Violet BW, C. I. No. 387, for staining either
animal or plant materials. Parenchyma is generally red to reddish
violet with the cell walls violet.

410. Chrysamine G is an acid dye, light 5. Stained tissues
are too fugitive to be of any value.

415. Pontachrome Orange R (Du Pont) is a direct dye, light
5. A neutral stain gives a fugitive orange on paraffin sections of
plant tissues. In an alkaline solution, blue-green algae are stained
a fugitive bright orange.

419. Erie Fast Red FD (NAC) is a direct dye, light 3 to 4.
All of the color is removed from stained paraffin sections upon
washing in water or in 95 per cent alcohol ; in this respect the dye
resembles Congo Red. In alkaline solutions blue-green algae are
deep red to reddish brown.

438. Trypan Red is a direct dye, light 4. A bright red is ob-
tained on plant tissues but the color is not very fast. Trypan Red
may be used as a vital stain.

STAIN TECHNIQUE 41

448. Benzopurpurin 4B is a direct dye, light 4 to 5. This dye
is less intense and more fugitive than Congo Red. It may be used
in vital staining.

450. Benzopurpurin B is a direct dye, light 4. Parenchyma
is stained a bright fugitive red.

463. Azo Blue is a direct dye, light 4. Azo Blue was the first
blue azo dye known ; it was seldom used because of the ugly reddish
shade produced on fabrics. For paraffin sections the color is too
fugitive. It is polychromatic, staining nuclei red and cytoplasm
blue. For polychromatic staining with a direct blue dye use
Niagara Sky Blue, C. I. No. 520.

477. Diamine Blue 3B, Trypan Blue is a direct dye, light 4.
Parenchyma is stained a bright blue; nuclei are sometimes tinted
red. It is used as a vital stain.

495. Benzopurpurin 10B is a direct dye, light 4 to 5. The
bright red on parenchyma is similar to the color produced by Congo
Red.

502. Benzoazurine G is a direct dye, light 4. Sometimes the
stain is polychromatic; then, the nuclei of parenchyma retain red;
the cytoplasm, blue to blue-violet. In green algae previously
treated with copper sulfate, spores are tinted orange-red ; the vege-
tative cells are dark blue or violet.

518. Niagara Sky Blue 6B is a direct dye, light 3; i gram in
95 ml. of distilled water, 5 ml. of 5 per cent sodium sulfate, and J
ml. of acetic acid. The two best direct blue dyes are Niagara Sky
Blue 6B and Niagara Sky Blue, C. I. No. 520. The former dye
cannot be used on tissues fixed with Gilson's solution, the latter can.
There is little difference in the appearance of the dye powders or
the solutions but the results obtained from the two stains are unlike.
The first dye is a shade lighter blue, requires two or three times as
long for staining, and, in combinations with Sun Yellow, C. I. No.
620, gives a clear brilliant green. For a contrast in green and red,
with tints of yellow and blue, use a mixture of nine parts Sun Yel-
low and one part Acid Fuchsine, on sections of animal or fungous
tissue for 10 to 30 minutes, and counterstain in Niagara Sky Blue
6B, two or three minutes. Niagara Sky Blue 6B should be tried

42 W. H. E M I G

on either animal or plant tissues using the combinations of stains
given under Niagara Sky Blue, C. I. No. 520.

520. Niagara Sky Blue, Direct Sky Blue is a direct dye, light
3 ; J gram in 95 ml. of distilled water, 5 ml. of 5 per cent sodium
sulfate, and J ml. of acetic acid. Niagara Blue 4B is a commercial
trade name originating from a former manufacturer of dyes.
Frequently it is necessary to employ a dye possessing basic prop-
erties, the - NH 2 radical, in order to produce a contrast with acid
dyes. Niagara Sky Blue supplies this need in many sections of
animal or fungous tissues where the structures are differentiated
more clearly than with Aniline Blue, C. I. No. 707. Suggestions
for the application of this blue counterstain are given under Sun
Yellow, C. I. No. 620, Orange II, C. I. No. 151, Crocein Scarlet
MOO, C. I. No. 252, Biebrich Scarlet, C. I. No. 280, and Acid
Fuchsine, C. I. No. 692.

On red and green algae, the polychromatic properties are evi-
dent; the spores are tinted red and the vegetative cells, blue to
violet. On mature plant tissues, the basic dyes such as Rhodamine
6G, C. I. No. 752, Acridine Red, C. I. No. 740, or Safranine, C. I.
No. 841, are followed by Niagara Sky Blue for one to five minutes.
Should the section consist largely of parenchyma, Biebrich Scarlet
and Niagara Sky Blue often supply a vivid contrast. On either
fungous tissues or meristems, the orange and red acid dyes such
as Orange II, Biebrich Scarlet, or Acid Fuchsine used for 30 min-
utes to 24 hours may be followed by Niagara Sky Blue one to five
minutes in the same manner as these combinations are used on ani-
mal materials.

Class V. Trisazo Colouring Matters

581. Erie Black GXOO, Chlorazol Black E, Direct Black E
is a direct dye. In the commercial dyeing of cotton, Erie Black is
applied at boiling temperature with the aid of a chromium salt. In
sections of embryos taken from a fixative containing chromium as
in chrom-acetic or Zenker's solution, the dye colors the nuclei black
and cytoplasm gray. The dye cannot be used as a general stain on
either animal or plant tissues. Parts of a pine needle section are
pale blue, dingy green, or dim yellow; on invertebrate and verte-

STAIN TECHNIQUE 43

brate tissues the unevenly distributed dull black is undesirable.
Specialized uses of Erie Black do not compare favorably with
Niagara Sky Blue, C. I. No. 520, or Haematoxylin, C. I. No. 1246.

593. Direct Green B, Diazine Green B is a direct dye, light 3
to 4. There are few green acid dyes suitable for animal tissues.
On invertebrates or paraffin sections, try Crocein Scarlet 7B, C. I.
No. 286, for five minutes and counterstain with Direct Green B
two minutes.

The spores of blue-green algae are blue-green or black; the
vegetative cells, clear blue. In an acid solution of the dye, green
or red algae are given a polychromatic stain that varies through
red to purple or blue. Parenchyma of vascular plants is dull blue.

594. Direct Green G, Alkali Green D is a direct dye, light 4 to
3 ; i gram in 95 ml. of distilled water, 5 ml. of 5 per cent sodium
sulfate, and 1 ml. of 4 per cent sodium hydroxide. This formula
constitutes the stain for blue-green algae and whole mounts giving
the best direct green ; it is moderately fast in glycerin. On paraffin
sections of animal tissues and vascular plants the dark green is too
indefinite.

Class VI. Tetrakisazo Colouring Matters

This class includes yellow, brown, and black direct dyes that are
not used as biological stains.

Chapter VI
BIOLOGICAL STAINS

A, Synthetic Organic Dyes (Continued)
Classes VII to XXVI

Class VII. Stilbene Colouring Matters

620. Sun Yellow is a direct dye, light 3 ; J gram in 95 ml. of
distilled water, 5 ml. of 5 per cent sodium sulfate, and £ ml. of
acetic acid. A few direct dyes react in a manner similar to a
mordant; such dyes are not always evident in the finished slide
but they assist in the fixation of the more important dye. Because
the color of Sun Yellow in combinations with blue dyes changes to
green, the relative amounts of yellow, green, and blue can be used
as an indicator to show when the proper degree of contrast has been
obtained. This dye on both animal and plant materials serves as
a mordant in the production of green in combinations with blue
counterstains ; this green is more fast to light than Fast Green
FCF, C. I. No. 670.

On whole mounts of invertebrates or on paraffin sections of
tissues, use Sun Yellow ten minutes; in mixtures with Orange II,
C. I. No. 151, the Crocein Scarlets, or Biebrich Scarlet, C. I. No.
280, for five minutes ; in mixtures with Acid Fuchsine, C. I. No.
692, try one to five minutes. The contrast dyes include : ( 1 ) Ani-
line Blue, C. I. No. 707, 15 to 30 seconds, (2) Niagara Sky Blue,
C. I. No. 520, 30 seconds to one minute, (3) New Methylene Blue,
C. I. No. 927, one minute, or (4) Erie Violet BW, C. I. No. 387,
five to eight minutes.

Use Sun Yellow or the mixtures containing Sun Yellow for 24
hours on mature plant tissues but ten minutes to an hour on meri-
stems. The counterstains require 15 seconds to five minutes de-
pending on the time given to the lighter stain.

44

STAIN TECHNIQUE

45

Combinations of Stains

Double stains
Sun Yellow

X

Triple stains

Sun Yellow (10) and
Orange II (5)

X

Sun Yellow (10) and
Crocein Scarlet MOO (5) X

Sun Yellow (10) and
Biebrich Scarlet (2)

Sun Yellow (10) and
Crocein Scarlet 7B (2)

X

X

Sun Yellow (10) and
Acid Fuchsine ( 1 )

X

Quadruple stains
Sun Yellow (10),
Orange II (5), and
Crocein Scarlet MOO (5) X

Sun Yellow (10),
Orange II (5), and
Biebrich Scarlet (2)

Sun Yellow (10),
Orange II (5), and
Acid Fuchsine (1)

.. X

X

Counterstains
Aniline Blue
New Methylene Blue
Niagara Sky Blue

New Methylene Blue
Niagara Sky Blue

Aniline Blue

New Methylene Blue

Niagara Sky Blue

New Methylene Blue
Niagara Sky Blue

Aniline Blue
New Methylene Blue
Niagara Sky Blue
Direct Green B
Erie Violet BW

Aniline Blue
New Methylene Blue
Niagara Sky Blue
Niagara Sky Blue 6B

New Methylene Blue
Niagara Sky Blue

Aniline Blue

New Methylene Blue

Niagara Sky Blue

Aniline Blue

New Methylene Blue

Niagara Sky Blue

46 W. H. E M I G

622. Mikado Yellow G, Stilbene Yellow is a direct dye, light
3. The stain is lighter than Sun Yellow but it is similar in other
respects.

Class VIII. Pyrazolone Colouring Matters

640. Tartrazine is an acid dye, light 4. The bright yellow dye
is valued for the coloring of foodstuffs, for light filters, or for
orthochromatic photographic plates.

Class IX. Ketonimine Colouring Matters

This class includes Auramine, C. I. No. 655, a basic dye that is
too fugitive for use on permanent slides.

Class X. Triphenylmethane and Diphenylnaphthylmethane Col-
ouring Matters

Although the first basic dyes were made from aniline, very few
of the coal-tar dyes originate from that source, either directly or
indirectly. Because the early basic dyes were fugitive there arose
a popular impression that all synthetic dyes were likewise deficient
in fastness. Many of these dyes exhibit the phenomenon of sur-
face color to a marked degree. Basic Fuchsine, C. I. No. 677, is
red when dissolved in water; the crystals have a beautiful beetle
green surface which is complementary in color to that of the solu-
tion. Because the basic stains in pure water readily undergo a
partial hydrolysis of the dye salt, they are dissolved in distilled
water with acetic acid. On exposure to sunlight, all green basic
dyes fade rapidly; this is attributed partly to the greater absorp-
tion of light rays by green pigments.

657. Malachite Green, China Green, Diamond Green, Solid
Green is a basic dye, light 4; J gram in 95 ml. of distilled water, 5
ml. of 5 per cent ammonium alum, and \ ml. of acetic acid. Mala-
cite Green is frequently used on xylem and sclerenchyma where it
is better than Methyl Green, C. I. No. 684. For a contrast of red
and green, stain the sections of plant tissues first in Crocein Scar-
let MOO, C. I. No. 252, Biebrich Scarlet, C. I. No. 280, or Acid
Fuchsine, C. I. No. 692, and follow with Malachite Green for 12
to 15 minutes.

STAIN TECHNIQUE 47

658. Setoglaucine O is a basic dye, light 5. The dye is lighter
and less fast than Malachite Green, C. I. No. 657. It is used in
certain bacteriological media.

662. Brilliant Green, Ethyl Green is a basic dye, light 5.
This dye has the same staining properties as Setoglaucine O, C. I.
No. 658. It, too, is used in bacteriological media.

665. Alkali Green is an acid dye, light 5. The pale dull green
obtained with Alkali Green is one of the most fugitive.

670. Light Green SF, Acid Green G, Fast Green FCF is an
acid dye; light 5 ; J gram in 100 ml. of distilled water and 1 ml. of
1 per cent sulfuric acid. Plant tissues are stained a fugitive green
or light blue-green. Fast Green FCF, which is the darker, should
be used only on thick sections of vascular tissues. Mature plant
materials taken from one of the basic dyes, Rhodamine, C. I. No.
752, Basic Fuchsine, C. I. No. 677, or Safranine, C. I. No. 841, are
counterstained in Fast Green FCF for 12 to 15 minutes. More
useful green cytoplasmic stains that have been described are the
combinations of Sun Yellow, C. I. No. 620, with New Methylene
Blue, Aniline Blue, or Niagara Sky Blue. On invertebrates and
whole mounts of fungi or algae, Naphthol Green B, C. I. No. 5,
applied with the aid of ferric alum produces a green more fast and
better differentiated than Fast Green FCF. Naphthol Green B is
also the better contrast green after iron Haematoxylin.

A quadruple stain which includes Fast Green FCF may be
applied to vascular tissues. The first stain consisting of a mixture
of five parts Orange II and one part Fast Green FCF is used for
15 to 30 minutes. After a rinse in distilled water, the material is
counterstained in three parts Safranine and one part Methyl Violet
for 15 to 30 seconds. If more of the green is wanted, use the first
mixed stain longer than 30 minutes ; with a longer interval in Safra-
nine and Methyl Violet, the percentage of violet is increased.

676. Pararosaniline is a basic dye, light 2 ; 1/10 gram in either
95 ml. of distilled water or 95 ml. of 50 per cent alcohol, 5 ml. of 5
per cent ammonium alum, and J ml. of acetic acid. The fuchsia on
plant tissues is brighter than the bluish red obtained with Basic
Fuchsine, C. I. No. 677. The dye may be used in staining bacteria
or in formulae calling for Basic Fuchsine.

;■

V'

48 W. H. EMIG

676a. Aldehyde Green, Aniline Green, Benzaldehyde Green is
a basic dye, light 4 ; J gram in 95 ml. of distilled water, 5 ml. of 5 per
cent ammonium alum, and % ml. of acetic acid. As a counterstain,
use Aldehyde Green for 15 minutes after Biebrich Scarlet, C. I.
No. 280, Acid Fuchsine, C. I. No. 692, Crocein Scarlet MOO, C. I.
No. 252, or Orange II, C. I. No. 151. It is similar to Malachite
Green, C. I. No. 657, but on xylem and sclerenchyma the green is a
shade brighter.

677. Basic Fuchsine, Aniline Red, Diamond Fuchsine, Ma-
genta, Roseine is a basic dye, light 2 ; \ gram in 95 ml. of distilled
water, 5 ml. of 5 per cent ammonium alum, and J ml. of acetic acid ;
warm the mixture until the dye dissolves. Although Basic Fuch-
sine is more or less fugitive on fabrics, it is comparatively fast on
paraffin sections. It is used in certain bacteriological media and in
the staining of bacteria. Vascular plant tissues are stained an
intense fuchsia to reddish violet in 15 minutes ; the best counterstain
is Fast Green FCF, C. I. No. 670. For a good triple stain on free-
hand sections use a mixture of nine parts Orange II, C. I. No. 151,
and one part Benzo Pure Blue, C. I. No. 520, for one hour, and after
a rinse in distilled water, counterstain with Basic Fuchsine one
minute.

678. New Fuchsine, New Magenta is a dye similar to but more
soluble and more bluish red than Basic Fuchsine.

679. Dahlia, Hoffmann Violet, Iodine Violet, Red Violet 5R is
a basic dye, light 2 to 3 ; 1/10 gram in 95 ml. of distilled water, 5 ml.
of 5 per cent ammonium alum, and J ml. of acetic acid. The dye
may be used in any of the procedures described under Methyl Violet,
C. I. No. 680. A fast pigment is obtained if the material is stained
for an hour and differentiated in 95 per cent alcohol. Depending
on the tissue, the color varies from a reddish violet to dark violet or
black.

680. Methyl Violet B, 2B is a basic dye, light 2 to 3 ; 1/10 gram
in 95 ml. of distilled water, 5 ml. of 5 per cent ammonium alum, and
J ml. of acetic acid. On animal tissues, basic dyes have a limited
value ; nuclei and a few kinds of special glandular cells such as gob-
let cells of the intestine and mucous cells of the epidermis are colored
much darker than the nonspecialized cytoplasmic structures.

STAIN TECHNIQUE 49

When compared with other shades of Methyl Violet on the same
kind of plant tissues, the 2B shade is the best of the series. As a
counterstain after Acid Fuchsine, C. I. No. 692, Biebrich Scarlet,
C I. No. 280, Crocein Scarlet MOO, C. I. No. 252, or Orange II,
C. I. No. 151, Methyl Violet B may be used for from five seconds
to five minutes depending on the time taken for the first and lighter
color. An especially noteworthy triple stain for root, stem, and
leaf sections is obtained by using a mixture of two parts Orange II
and one part Biebrich Scarlet for 24 hours, and after a rinse in
distilled water, a counterstain in Methyl Violet B for two or three
minutes. The colors are very fast as well as brilliant.

681. Crystal Violet, Methyl Violet 10B is a basic dye, light 3.
Gentian Violet is either a mixture of two or more shades of Methyl
Violet or it is Crystal Violet containing dextrin. Crystal Violet is
more selective in its staining of minute cell structures than other
basic violet dyes. The dye is dissolved in distilled water with
ammonium alum and acetic acid ; it is used in the same manner as
and after the same acid dyes given in combinations with Methyl
Violet B.

682. Ethyl Violet is a basic dye, light 5. Tissues are not
stained uniformly and the color is very fugitive.

683. Methyl Violet 6B, Benzyl Violet, Gentian Violet 6B is a
basic dye, light 3 ; 1/10 gram in 95 ml. of distilled water, 5 ml. of 5
per cent ammonium alum, and ^ ml. of acetic acid. Plant tissues
are stained blue-violet similar to the color obtained with Crystal
Violet but the differentiation of minute cell structures is not so
clear. As a counterstain, Methyl Violet 6B is used for three min-
utes after most of the red acid dyes such as Acid Fuchsine, Crocein
Scarlet MOO, or Orange II.

684. Methyl Green is a basic dye, light 4. The Methyl Green
now on the market, both the domestic and imported product, is a
blue or blue-green dye ; it is unstable and fugitive. The green dye
of former manufacture, labeled Methyl Green, was a different dye
or a mixture. On various plant tissues the stain is blue-green to
blue-violet. Instead of Methyl Green use Methylene Green, C. I.
No. 924.

50 W. H. E M I G

686. Iodine Green is a basic dye, light 4. Different plant tis-
sues are stained blue-green or dull blue without a trace of green.
Similar to Methyl Green, it should be replaced by the better dye,
Methylene Green, C. I. No. 924.

689. Opal Blue, Aniline Blue alcohol soluble, Bleu Lumiere,
Gentiana Blue 6B, Spirit Blue is a basic dye, light 3 ; ^ gram in 95
ml. of 50 per cent alcohol, 5 ml. of 5 per cent ammonium alum, and
^ ml. of acetic acid. The cytoplasm is more readily stained an
intense blue than are the nuclei. Opal Blue may be used whenever
the color produced by New Methylene Blue is too dark. In con-
trast staining use Biebrich Scarlet, C. I. No. 280, Crocein Scarlet
MOO, C. I. No. 252, or Orange II, C. I. No. 151, for 24 hours and
Opal Blue five minutes ; transfer the section from the stain directly
to 95 per cent alcohol for dehydration.

692. Acid Fuchsine, Acid Azo Fuchsine, Acid Magenta, Acid
Rubine, Fuchsine S, Rubine S is an acid dye, light 2 to 3 ; i gram
in 100 ml. of distilled water and 1 ml. of 1 per cent sulfuric acid.
The dye sold previous to 1920 was a fugitive red dye, probably
Azorubine ; that now on the market, domestic or imported, if true
to name, gives a fuchsia to reddish violet of good fastness. By the
sulfonation of different Basic Fuchsines, various quantities of the
derived acid dye are red to violet depending on the particular basic
dye used in its manufacture. If a bright red is not obtained with
Acid Fuchsine, use Biebrich Scarlet, C. I. No. 280, on plant tissues
or Crocein Scarlet MOO, C. I. No. 252, on animal tissues.

In the staining of animal tissues, Acid Fuchsine usually blends
with blue or violet dyes to give colors of little contrast. If Acid
Fuchsine is diluted with either Sun Yellow, C. I. No. 620, or Orange
II, C. I. No. 151, the contrast with blue stains will be brilliant. A
good combination of stains for whole mounts, invertebrates, chick
embryos, or paraffin sections of tissues includes a mixture of ten
parts Sun Yellow and one part Acid Fuchsine for five minutes and
a counterstain either in Aniline Blue, C. I. No. 707, 15 seconds, or
in Niagara Sky Blue, C. I. No. 520, 30 seconds. Another good
technique includes a mixture of ten parts Orange II and one part
Acid Fuchsine for one or two minutes and a counterstain in Aniline
Blue or Niagara Sky Blue.

STAIN TECHNIQUE 51

In staining mosses, ferns, or seed plants, use a mixture of
twenty parts Sun Yellow and one part Acid Fuchsine for 24 hours ;
counterstain in New Methylene Blue, C. I. No. 927, five minutes.
The basic dye adds a clear outline to the cell walls. Because fun-
gous tissues are similar to animal tissues in their reactions to dyes,
the combinations given in the previous paragraph should be used.
Double stains on vascular plants require Acid Fuchsine for 1 5 min-
utes or longer and a counterstain : ( 1 ) Aldehyde Green, C. I. No.
676a, 15 minutes, (2) Methyl Violet B, C. I. No. 680, one to three
minutes, (3) Methylene Green, C. I. No. 924, 15 minutes, (4)
Victoria Blue B, C. I. No. 729, five minutes, (5) Thionine, C. I.
No. 920, five minutes, or (6) Opal Blue, C. I. No. 689, one to ten
minutes.

698. Acid Violet is an acid dye, light 4. The dye sold under
this name is of variable composition. Because plant tissues are not
stained uniformly, a good acid violet may be obtained with Erie
Violet BW, C. I. No. 387 ; sometimes Acid Fuchsine, C. I. No. 692,
is satisfactory.

703. Alkali Blue 6B is an acid dye, light 4 to 5. On paren-
chyma a fugitive blue to blue-green provides little of value for per-
manent slides. In alkaline solutions of the dye, blue-green algae
become clear bright blue, but, unfortunately, the color is not fast.

705, 706, and 707. Aniline Blue water soluble, China Blue,
Cotton Blue, Methyl Blue, Soluble Blue, Water Blue are closely
related acid dyes, light 3 ; J gram in 95 ml. of distilled water, 5 ml.
of 5 per cent ammonium alum, and 1 ml. of 1 per cent sulfuric acid.

On animal tissues, Aniline Blue and Niagara Sky Blue, C. I.
No. 520, comprise an important pair of contrast stains. Should
the former of these blue dyes not provide a clear differentiation of
tissues, the latter usually supplies the desired quality. In most
combinations of stains including Orange G, C. I. No. 27, Orange
II, C. I. No. 151, Orange R, C. I. No. 161, Crocein Scarlet MOO,
C. I. No. 252, or Sun Yellow, C. I. No. 620, Aniline Blue requires
15 seconds to one minute. Aniline Blue is also a constituent in
several triple stains which are described under Sun Yellow, Orange
II, and Crocein Scarlet MOO.

52 W. H. E M I G

Aniline Blue is of greater importance on plant tissues than on
animals. After a 24-hour stain in a basic dye such as Rhodamine
6G, C. I. No. 752, Acridine Red, C. I. No. 740, or Safranine, C. I.
No. 841, the best blue counterstain is Aniline Blue for five min-
utes.

710. Isamine Blue 6B is an acid dye, light 3 to 4. The color
and staining properties of the dye are similar to those of Aniline
Blue ; it is less fast.

712. Acid Blue G, Brilliant Acid Blue V is an acid dye, light 5.
Parenchyma is stained a light blue-green that is lacking in defini-
tion of details and in fastness.

714. Patent Blue A, Brilliant Acid Blue A is an acid dye, light
4. Parenchyma is stained blue-green but the details of structure
are not presented clearly.

720. Eriochrome Azurol V is a mordant dye, light 4 to 3 ; J
gram in 90 ml. of distilled water, 5 ml. of J per cent potassium
dichromate, and 5 ml. of 5 per cent sodium sulfate. Cell walls of
parenchyma are stained a clear blue and the cytoplasm, violet ; the
nuclei remain unchanged. The color is similar to that obtained
with Niagara Sky Blue.

724. Aurin is the free acid Rosolic Acid used as an indicator.
Coralline, the sodium salt of Rosolic Acid, is used for the manufac-
ture of turkey red lakes.

729. Victoria Blue B is a basic dye, light 3 ; J gram in 95 ml.
of distilled water, 5 ml. of 5 per cent ammonium alum, and J ml.
of acetic acid. A clear blue is obtained in parenchyma or scleren-
chyma. Sometimes this stain gives better contrasts with red acid
dyes than New Methylene Blue, C. I. No. 927. As a counterstain
use Victoria Blue B after Acid Fuchsine, C. I. No. 692, Biebrich
Scarlet, C. I. No. 280, Crocein Scarlet MOO, C. I. No. 252, or
Orange II, C. I. No. 151.

731. Night Blue is a basic dye, light 4. The beautiful blue-
violet obtained on plant tissues is not very fast to light.

736. Acid Blue B is an acid dye, light 5. Tissues stained with
this dye are not clearly differentiated ; they are too light and very
fugitive.

STAIN TECHNIQUE 53

Class XL Xanthene Colouring Matters

The majority of the Xanthene dyes are characterized by the
strong fluorescence of their solution which is attributed to the pres-
ence of the pyrone ring in the dye molecule.

739. Pyronine 2G is a basic dye, light 1 to 2 ; £ gram in 95 ml.
of distilled water, 5 ml. of 5 per cent ammonium alum, and \ ml. of
acetic acid. Plant tissues are stained a bright red to fuchsia. The
color is similar to that of Basic Fuchsine, C. I. No. 677, but Pyro-
nine 2G is more easily differentiated in double staining than Basic
Fuchsine. The best contrast is obtained with either Niagara Sky
Blue, C. I. No. 520, or alum Haematoxylin. For staining yeast
spores or bacteria use 5 per cent of the dye instead of the ^ gram
in the regular formula. A film of yeast containing spores is dried
on a slide, left in 5 per cent chromic acid for 30 minutes, rinsed with
distilled water, stained in Pyronine 2G at 50° C. for one hour, and
counterstained in Niagara Sky Blue for five minutes.

740. Acridine Red is a basic dye, light 2 ; ^ gram in 95 ml. of
distilled water, 5 ml. of 5 per cent ammonium alum, and J ml. of
acetic acid. Plant tissues are pink to bright red. It may be used
in any technique where Safranine, C. I. No. 841, gives good results
but the color will be brighter than that of Safranine. A section
taken from this stain may be counterstained in Aniline Blue, C. I.
No. 707, Niagara Sky Blue, C. I. No. 520, or alum Haematoxylin,
C. I. No. 1246.

741. Pyronine B is a basic dye with properties similar to those
of Pyronine 2G; it is more bluish red.

749. Rhodamine B, 750. Rhodamine G, and 752. Rhoda-
mine 6G are basic dyes, light 2 ; ^ gram in 95 ml. of distilled water,
5 ml. of 5 per cent ammonium sulfate, and J ml. of acetic acid.
Rhodamine 6G is a bright pink ; it is more easily differentiated with
blue acid or direct dyes than any other red basic stain. Rhodamine
6G for 12 to 24 hours does not overstain paraffin sections, freehand
sections of vascular plants, or woody materials. The counterstains
include: (1) Aniline Blue, C. I. No. 707, five minutes, (2) Niagara
Sky Blue, C. I. No. 520, five minutes for red and light blue, or (3)
alum Haematoxylin, C. I. No. 1246, for bright red and purple.

54 W. H. E M I G

764. Phenolphthalein is used extensively as an indicator.

766. Fluorescein, Uranine is an acid dye, light 5 to 4. Fluo-
rescein does not stain plant tissues unless potassium dichromate is
present, whereupon, a yellow to brown is obtained which is equal to
Eosin in fastness.

768. Eosin Y, 769. Methyl Eosin, 770. Ethyl Eosin, and
771. Eosin B are acid dyes, light 4 to 5. Few dyes are so easily
precipitated by acids as Eosin and Erythrosin. They are fre-
quently used in counterstaining bacteria, fungi, animal tissues, or
whole mounts. The fugitive nature of Eosin does not recommend
its use for permanent preparations. By adding potassium dichro-
mate to the stain, the fastness is improved slightly and the color is
changed to a darker tint.

772. Erythrosin G, Y, and 773. Erythrosin B are acid dyes,
light 4 to 5 ; £ gram in 100 ml. of distilled water with or without
the addition of 5 ml. of \ per cent potassium dichromate. Potas-
sium dichromate increases the fastness and changes the color to
bright scarlet. Erythrosin is used as a food color. On animal tis-
sues Erythrosin usually blends with other dyes to yield colors of
little contrast. A fairly good differentiation occurs with a nuclear
stain in Haematoxylin followed by Erythrosin or ten minutes in
Erythrosin followed by Niagara Sky Blue, C. I. No. 520, for two
minutes. Instead of Erythrosin, try Crocein Scarlet MOO, C. I.
No. 252.

Sections of plant tissues are stained one to ten hours in Ery-
throsin and then counterstained with Capri Blue, C. I. No. S76,
about five minutes, for pink and blue. This combination of stains
will accomplish results that are impossible with Cyanine, C. I. No.
806, and Magdala Red, C. I. No. 857.

774. Phloxine B, 776. Cyanosine, 777. Rose Bengale, 778.
Phloxine B, 779. Rose Bengale B, and 780. Cyanosine B are
similar to Erythrosin in color and staining properties but they are
more fugitive. The addition of potassium dichromate to the stain
does not improve the fastness.

781. Gallein is a mordant dye, light 1 ; J gram in 100 ml. of
either 1 per cent ammonium acetate or 1/10 per cent sulfuric acid.

STAIN TECHNIQUE 55

Small invertebrates are mordanted for 30 minutes in 1 per cent
ferric ammonium sulfate. After a rinse in distilled water, the
material appears blue-black after one or two minutes in the stain
at 50° C. With copper acetate as the mordant the color is Hema-
tein-purple. Paraffin sections of animal tissues are stained violet
with blue-black nuclei in 15 to 30 seconds at 50° C.

Paraffin sections of plant tissues mordanted in ferric alum and
kept for five to ten minutes in the dye at 50° C. become reddish vio-
let in the cytoplasm to blue-violet in the nuclei. With this dye in
dilute sulfuric acid, the sections mordanted in iron alum take a blue
nuclear stain. Whole mounts take on a Hematein-purple of ex-
cellent fastness. Blue-green algae are readily stained without the
aid of a mordant.

783. Cerulein MS, Anthracene Green, Coerulein MS is a mor-
dant dye, light 3 to 4; J gram in 100 ml. of 1 per cent ammonium
acetate. This dye does not give a satisfactory stain on animal tis-
sues. Vascular plant tissues mordanted in ferric alum become dull
green in the stain at 50° C. In alkaline solutions of the dye blue-
green algae are clear green.

Class XII. Acridine Colouring Matters

785. Acridine Yellow R, 2R is a basic dye, light 2; £ gram in
95 ml. of distilled water, 5 ml. of 5 per cent ammonium alum, and ■£■
ml. of acetic acid. Unless the stain contains ammonium alum, the
dye bleeds from sections mounted in balsam ; more than the usual
amount of time is needed for dehydration. The dye may be used
in preference to Bismarck Brown, C. I. No. 331.

788. Acridine Orange is a basic dye, light 1 to 2 ; £ gram in 95
ml. of distilled water, 5 ml. of 5 per cent ammonium alum, and \ ml.
of acetic acid. Plant tissues are stained a clear brown or dark
orange of exceptional fastness. To prevent bleeding of the color
in balsam, dehydrate the material slowly.

793. Phosphine is a basic dye, light 2 to 3. This dye is a by-
product obtained in the manufacture of Basic Fuchsine. The stain
is similar in color to Acridine Orange but not of equal fastness.
The dye-solution decomposes on standing.

56 W. H. E M I G

Class XIII. Quinoline Colouring Matters

A number of dyes with the property of color-sensitizing photo-
graphic plates belong here. Most of them are fugitive and ex-
pensive.

801. Quinoline Yellow is an acid dye, light 2 ; J gram in 95 ml.
of distilled water, 5 ml. of £ per cent potassium dichromate, and 1
ml. of 1 per cent sulfuric acid. On most plant tissues this stain is
too light. A clear bright yellow is obtained more easily with the
addition of potassium dichromate.

806. Cyanine is a basic dye, light 5. It is one of the most ex-
pensive as well as one of the most fugitive dyes. Nuclei are stained
blue ; the cytoplasm and cell walls have only a faint color. Algae in
whole mounts are not stained evenly. The same color and stain-
ing reactions can be obtained with Capri Blue, C. I. No. 876.

Class XIV. Thiazole and Thiobenzenyl Colouring Matters

To this class belongs Primuline, C. I. No. 812, which is a fugi-
tive acid dye that has little value for histological purposes.

Class XV. Indamine Colouring Matters

Indamine dyes are of no value as stains ; they are intermediate
products used in the manufacture of sulfur dyes.

Class XVI. Indophenole Colouring Matters

These substances are used in the manufacture of vat dyes.

Class XVII. Azine Colouring Matters

825. Neutral Red, Toluylene Red is a basic dye, light 3. Plant
tissues stained a dull red in acid solutions become more intense red
with the addition of ammonium alum. This dye is frequently used
as a vital stain ; the various parts of the living cell are differentiated
in orange or red. It is also used as an indicator.

826. Neutral Violet is a basic dye, light 4. Plant tissues are
stained a fugitive blue-violet. The dye solution decomposes on
standing.

828. Azocarmine GX is an acid mordant dye, light 3 to 4.
The dye dissolves in either 1 per cent ammonium acetate or in 5 per

STAIN TECHNIQUE 57

cent ammonium alum. Invertebrates are orange-red after 15 min-
utes at 50° C. if ferric alum or copper acetate is used as the mor-
dant. On most paraffin sections the color is too light. For a gen-
eral stain of this color try Direct Fast Scarlet 4BS, C. I. No. 327.

841. Safranine A, G, O, Y is a basic dye, light 2 to 3; J gram
in 95 ml. of distilled water, 5 ml. of 5 per cent ammonium alum, and
J ml. of acetic acid. The letters Y (yellow) and G (gelb) refer to
the bright scarlet shade ; A and O indicate a product of the highest
purity or of a greater dye content. Safranine is soluble in either
water or alcohol.

Safranine, without the addition of an acid, stains slowly; the
color washes out during dehydration. The presence of ammonium
alum intensifies the color and reduces the time required for stain-
ing. Safranine and alum Haematoxylin is one of the favorite com-
binations for leaf, stem, and root sections ; here the lack of contrast
between red and purple gives blue or green dyes a preference to
Haematoxylin. The material is stained in Safranine long enough
to obtain an intense red, one to ten hours, and then, without destag-
ing, the contrast color is added. The best counterstains for Safra-
nine are : ( 1 ) Aniline Blue, C. I. No. 707, five minutes, (2) Niagara
Sky Blue, C. I. No. 520, five to ten minutes, (3) Fast Green FCF,
C. I. No. 670, ten to 15 minutes, or (4) alum Haematoxylin. After
the material is stained in alum Haematoxylin a rinse in 0.01 per cent
ammonium hydroxide will darken the purple pigment. A discus-
sion of Flemming's triple stain, Safranine, Orange G, and Gentian
Violet is given under Orange G, C. I. No. 27.

842. Fuchsia, Methylene Violet is a basic dye, light 3. Fuchsia
has properties similar to Safranine; the color of stained tissues is
more bluish and varies from fuchsia to reddish violet.

846. Mauveine is a basic dye, light 3. Mauveine, the first dye
made from aniline, was discovered in 1856. Paraffin sections of
plant tissues are stained blue-violet similar to the color obtained
with Methyl Violet. »

856. Naphthyl Red is a basic dye, light 5. Only the nuclei of
mature plant cells are stained a fugitive red.

857. Magdala Red is a basic dye, light 3 ; 1/10 gram in 100 ml.

58 W. H. E M I G

of 50 per cent alcohol and J ml. of acetic acid. The nuclei of paren-
chyma are stained bright red ; the cytoplasm and cell walls, fuchsia.

860. Acetin Blue R, Induline alcohol soluble, is a basic dye,
light 4. Plant tissues in paraffin sections are stained a dull light
blue.

861. Induline water soluble is an acid dye, light 4 to 5. Plant
tissues are stained light blue in acid solutions of the dye or black
with the addition of potassium dichromate.

865. Nigrosine water soluble is an acid dye, light 3 to 4. Pa-
renchyma is stained dull blue in an acid solution of the dye. With
potassium dichromate present the cell walls are blue ; the nuclei re-
main unchanged or yellow-brown.

Class XVIII. Aniline Black and Allied Colouring Matters
Class XIX. Oxazine Colouring Matters

876. Capri Blue is a basic dye, light 3 ; 1/10 gram in 95 ml. of
distilled water, 5 ml. of 5 per cent ammonium alum, and -J ml. of
acetic acid. Plant tissues are stained blue or black. This dye may
be used in preference to Cyanine, C. I. No. 806; it should counter-
stain well after Erythrosin, C. I. No. 773.

877. Brilliant Cresyl Blue, Brilliant Blue is a basic dye, light
3. Certain plants in whole mounts are stained blue but the color is
not of uniform density.

883. Gallocyanine is a direct mordant dye. The pigment ob-
tained at room temperature is fugitive, light 4. At 50° C. the color
is intensified to a deep blue-black with an increase in fastness to that
of the best dyes, light 1 ; 1/10 gram in 100 ml. of 1 per cent am-
monium acetate.

Invertebrates are placed in 1 per cent ferric ammonium sulfate
for 30 minutes. After a rinse in distilled water, the material, at
50° C, becomes a Hematein-purple in one or two minutes. Paraffin
sections of animal tissues are violet within 30 seconds. Plant mate-
rials mordanted in ferric alum require five to ten minutes for whole
mounts or 15 to 20 minutes on paraffin sections.

909. Meldola Blue is a basic dye, light 4 to 5. The dye is
somewhat selective in that the nuclei are stained blue-black and the

STAIN TECHNIQUE 59

cytoplasm blue-violet. With the addition of ammonium alum to the
stain the color on plant tissues is more intense but it is still too fugi-
tive to be of much value in histology.

913. Nile Blue Sulfate is a basic dye, light 2. The dye is
slightly soluble in warm water but readily soluble in 50 per cent
alcohol. It is unsuited for paraffin sections. Green algae which
are usually overstained in basic dyes are colored a clear bright blue.
The dye may be used as a vital stain.

917. Alizarine Green G is an acid mordant dye, light 1 ; 1/10
gram in 100 ml. of 1 per cent ammonium acetate. With 1 per cent
ferric alum as a mordant followed by the stain for 30 minutes at
50° C. the resultant green is the clearest obtained with a mordant
dye. Paraffin sections of animal tissues are stained green slightly
tinted blue. Various plant tissues appear dull green with the am-
monium acetate solution or blue in the stain containing sulfuric acid.
Alizarin Green G may be used as a vital or as a fat stain.

Class XX. Thiamine Colouring Matters

920. Thionine is a basic dye, light 3; 1/10 gram in 95 ml. of
distilled water, 5 ml. of 5 per cent ammonium alum, and J ml. of
acetic acid. Plant tissues are stained dark blue to violet. In those
examples of contrast staining where Methyl Violet does not differ-
entiate well, Thionine may give the desired results. Blue-green
algae are stained purple in alkaline solutions of the dye but this color
is not fast in glycerin. As a counterstain, Thionine may be used
after Acid Fuchsine, C. I. No. 692, Biebrich Scarlet, C. I. No. 280,
or Orange II, C. I. No. 151.

922. Methylene Blue is a basic dye, light 2 to 3 ; J gram in 95
ml. of distilled water, 5 ml. of 5 per cent ammonium alum, and h ml.
of acetic acid. Unna's polychrome Methylene Blue is Methylene
Blue ripened in a solution containing potassium carbonate. The
color of the dye is fugitive unless an acid and ammonium alum are
added to the dye solution. This dye has been used more than any
other basic blue dye for staining bacteria, blood, and animal tissues.
Because blood stains provide only temporary mounts, the technique
has been omitted. New Methylene Blue is more soluble ; for plant
tissues it is a better stain than Methylene Blue.

60 W. H. E M I G

923. Methylene Azure, Azure I is a basic dye, light 3; 1/10
gram in 95 ml. of distilled water, 5 ml. of 5 per cent ammonium
alum, and £ ml. of acetic acid. Methylene Blue in solution changes
to Methylene Azure by oxidation. Methylene Azure includes a
mixture of Azure A and Azure B. Azure C is another oxidation
product of Methylene Blue. Azure II is a mixture of equal parts
of Methylene Blue and Methylene Azure. All of these dyes are
polychromatic, a property which makes them valued for blood
stains. The preparation of blood stains includes the oxidation of
Methylene Blue and the addition of eosin.

By staining a section of pine wood in Methylene Azure or
Methylene Blue for 15 minutes, the torus becomes bright red while
the walls of the tracheids are blue; by staining for a longer time
only a blue is obtained. Unfortunately, these colors are not fast.
The addition of acids, alkalies, or ammonium alum greatly affects
the stain. Acid solutions of the dye react slowly with a better dif-
ferentiation than neutral solutions; an alkaline solution will stain
25 times more rapidly than an acid solution. The addition of am-
monium alum intensifies the color in either dye-solution.

924. Methylene Green is a basic dye, light 3 ; £ gram in 95 ml.
of distilled water, 5 ml. of 5 per cent ammonium alum, and -J ml.
of acetic acid. In acid solutions of the dye, plant tissues are stained
green to blue-green. The intensity of the color is reduced but more
clear in differentiation if ammonium alum is present in the stain.
This dye is the best of the green basic dyes; it provides a good
counterstain after Acid Fuchsine, C. I. No. 692, Biebrich Scarlet,
C. I. No. 280, or Crocein Scarlet MOO, C. I. No. 252.

925. Toluidine Blue O is a basic dye, light 3. Plant tissues
become blue similar in most respects to Methylene Blue. It may be
used as a substitute for Thionine, C. I. No. 920, or Azure A, C. I.
No. 923.

926. Rhoduline Blue GO, Thionine Blue GO is a basic dye,
light 4. Plant tissues are stained blue or blue-green that is similar
to but not so fast as Methylene Blue, C. I. No. 922.

927. New Methylene Blue, N, NSS is a basic dye, light 2 to 3 ;
J gram in 95 ml. of distilled water, 5 ml. of 5 per cent ammonium

STAIN TECHNIQUE 61

alum, and i ml. of acetic acid. New Methylene Blue is a constituent
of several triple and quadruple stains which are described under the
separate acid dyes. These combinations of stains can be used on
sections of embryonic animal tissues. For the general staining of
plant tissues New Methylene Blue gives better results than either
Methylene Blue or Methylene Azure. It may be used as a contrast
stain after Acid Fuchsine, C. I. No. 692, Biebrich Scarlet, C. I. No.
280, Crocein Scarlet MOO, C. I. No. 252, Orange II, C. I. No. 151,
or Sun Yellow, C. I. No. 620.

931. Brilliant Alizarin Blue is a basic dye, light 3 to 4. This
stain is darker than New Methylene Blue. The stain is not so good
with the addition of ammonium alum ; with alkalies very little color
is produced.

Class XXI. Sulfur and Sulfide Colouring Matters

Many of the sulfur dyes are not classified in the Colour Index
because their chemical composition at present is unknown. There
are no bright red sulfur dyes.

948. Sulfur Yellow, or Sulfur Orange, and 949. Sulfur
Brown 4G differ very little from Sulfur Bordeaux.

956. Sulfur Direct Blue, Sulfogene Direct Blue BRS (Du
Pont) is a direct dye, light 2; 1/10 gram in 95 ml. of 1 per cent
sodium sulfide and 5 ml. of 4 per cent sodium hydroxide. Blue-
green algae are not stained so intensely nor so brightly as with
Sulfur Brilliant Blue. The details of cell structure are presented
very clearly.

957. Sulfur Brilliant Blue, Sulfindone Brilliant Blue CG
(NAC), Sulfogene Brilliant Blue 6BS (Du Pont), and Sulfogene
Brilliant Blue 3GCF (Du Pont) are direct dyes, light 2 ; 1/10 gram
in 95 ml. of 1 per cent sodium sulfide and 5 ml. of 4 per cent sodium
hydroxide. Because the stain does not keep over a day, it must be
made as needed. These are the best blue sulfur dyes ; the color is
nearly violet on algae mounted in glycerin. Algae are placed in the
stain one to three hours at 50° C, rinsed with tap water, and then
oxidized to a deeper shade as soon as the moist plants are exposed
to air, for 15 minutes. Invertebrates such as Obelia, or jelly-fish

62 W. H. E M I G

of large size are stained within 15 minutes. All of these materials
are dehydrated from 10 per cent glycerin.

1006. Sulfur Green, Sulfogene Green 2B (Du Pont), Sulfo-
gene Brilliant Green 2G (Du Pont), and Sulfur Green 3G cone.
(NAC) are direct dyes, light 2; 1/10 gram in 100 ml. of 1 per cent
sodium sulfide and 1 ml. of 4 per cent sodium hydroxide. The 3G
shade gives a bright green; the 2B, a blue-green at 50° C. on blue-
green algae in three to ten hours, on invertebrates in 30 minutes.

1012. Sulfur Bordeaux, Sulfogene Bordeaux BRN (Du
Pont), and Sulfur Bordeaux BCF (NAC) are direct dyes, light 2;
1/10 gram in 95 ml. of 1 per cent sodium sulfide and 5 ml. of 4 per
cent sodium hydroxide. At 50° C., plant tissues are stained in 12
to 24 hours, invertebrates in 30 minutes. The oxidation treatment
is unnecessary for Sulfur Bordeaux.

Class XXII. Hydroxy ketone Colouring Matters
These dyes are used in the color printing of wool.

Class XXIII. Anthraquinone Colouring Matters

1027. Alizarin is an acid mordant dye, light 4 to 3; ^ gram in
100 ml. of distilled water and 1 ml. of 4 per cent sodium hydroxide.
The dye is insoluble in water, slightly soluble in alcohol, and blue-
violet in dilute alkalies. Upon the addition of an acid to the alka-
line stain, a yellow precipitate is formed. The dye produces a wide
range of colors depending on the metallic salt used as a mordant, the
alkalinity of the stain, and the temperature of application. A few
of the mordants with the accompanying color lakes are : aluminum,
orange to red ; copper, carmine ; antimony, brown ; chromium, bor-
deaux ; uranium, blue-violet ; and iron, blue-black to black. All of
the lakes are darker when developed at 50-60° C.

The stain is easily applied to invertebrates of any size. The
technique includes a mordant for 30 minutes ; after a rinse in dis-
tilled water, Alizarin is applied at 40-50° C. for five minutes. The
best colors develop after : ammonium alum, orange-red to red ; cop-
per acetate, carmine ; or ferric alum, dark red at room temperature
to blue-black at 50° C. Paraffin sections of earthworm are dull red

STAIN TECHNIQUE 63

in one minute when ferric alum is used as the mordant but the color
is not clear and the differentiation is not satisfactory.

A good brick red may be obtained on plant tissues with the fol-
lowing procedure: 15 minutes in turkey red oil solution, rinse in
distilled water, 15 minutes in a saturated solution of ammonium
alum, rinse in water, five minutes in lime water, rinse, and 12 to 24
hours in an alkaline solution of Alizarin. Turkey red oil is sul-
fonated castor oil. To 100 ml. of distilled water is added 1 ml. of
turkey red oil and two or three drops of ammonium hydroxide to
give a clear solution.

1034. Alizarin Red S, Alizarin Sodium Sulfonate is a direct
mordant dye, light 4 ; ^ gram in 95 ml. of distilled water, 5 ml. of 5
per cent ammonium alum, and \ ml. of acetic acid. After a plant
section has been in the stain 12 to 24 hours, it is rinsed in distilled
water and dipped into turkey red oil solution in order to develop the
orange-red pigment.

1037. Purpurin is an acid mordant dye, 3 to 4; 1/10 gram in 80
ml. of 5 per cent ammonium alum, boil, cool, filter, and add 20 ml. of
95 per cent alcohol which serves as a preservative. Plant tissues
are stained a brilliant red within 24 hours. The dye counterstains
easily with a large number of basic blue, violet, or green dyes. Un-
fortunately, this red is fugitive.

1048. Acid Alizarin Blue GR is a direct mordant dye, light 2.
The dye dissolves either in 1 per cent ammonium acetate or in 5 per
cent ammonium alum. The stain is not active until heated to 50°
C. ; at this temperature, mordanted sections of plant tissues are
stained bright blue. It is difficult to obtain a satisfactory prepara-
tion because a precipitate separates from solution soon after the
stain is heated.

1049. Acid Alizarin Green G is a direct mordant dye, light 1 ;
1/10 gram in 100 ml. of 1 per cent ammonium acetate. Blue-green
and green algae or fungi mordanted in ferric alum become clear
green within five to ten minutes at 50° C. The dye is not so good
for invertebrates in whole mounts. On paraffin sections of animal
tissues mordanted in 1 per cent ferric alum, light green is produced
in 30 seconds and clear green in one minute at 50° C.

Ay • f

/

64 W. H. E M I G

1050. Alizarin Cyanine R is an acid mordant dye. A solution
of this dye is not stable and, on heating, a reddish precipitate pre-
vents the development of a clear stain.

1051. Alizarin Cyanine G is a direct mordant dye, light 1;
1/10 gram in 100 ml. of 1 per cent ammonium acetate. Sections of
plant materials that are mordanted in ammonium alum are stained
a clear Hematein-purple. Objects that are mordanted in ferric
alum are red-violet to blue-violet within five to ten minutes at 50° C.
Blue-green algae are stained purple in alkaline solutions. When
ferric alum is used as a mordant, paraffin sections of animal tissues
or whole mounts of invertebrates are blue-violet in two to three
minutes at 50° C.

1054. Alizarin Sapphire BN (NAC) is a direct mordant dye,
light 2. Sections mordanted in ferric alum show only a trace of
color at 50° C. in the ammonium acetate strain ; in the sulfuric acid
solution of the dye, nuclei are light blue.

1067. Alizarin Blue S is a mordant dye, light 3. The dye dis-
solves in slightly alkaline 50 per cent alcohol but the blue pigment
separates from solution either in the presence of acids, alkalies, or
upon heating.

Class XXIV. Anthraqninone Vat Colouring Matters

1101. Car banthrene Jade Green (NAC).

1104. Carbanthrene Violet 2R (NAC).

1113. Carbanthrene Blue GD (NAC).

1131. Ponsol Red 5GK (Du Pont).

Ponsol Red AFF (Du Pont) .

Carbanthrene Brilliant Orange RK (NAC).

1162. Carbanthrene Red BN (NAC).

Sulfanthrene Red 3B (Du Pont).

A few of the cold vat dyes may be applied to blue-green algae at
50-60° C. The preparation of the stain is described under Indigo,
CI. No. 1177.

Class XXV. Arylidoquinone Vat Colouring Matters
Class XXVI. Indigoid Colouring Matters

1177. Indigo is a vat dye, light 1; 1/10 gram of the dye is

STAIN TECHNIQUE 65

worked into a paste in a casserole with 5 ml. of 35 per cent sodium
hydroxide, to which is added slowly 45 ml. of hot water, and then,
with continuous stirring, ^ gram of sodium hydrosulfite powder.
The vat may be colorless or it may be different from the original
dye; Indigo changes from blue to yellow-green in the vat. After
five to ten minutes at 60° C, the material is taken out of the stain
and rinsed in warm water. The stained object should be the same
color as the original dye. Pigments obtained from bright orange
or red dyes disappear from blue-green algae soon after the algae
are placed in glycerin, but jade green, violet, and indigo are fast
colors.

1180. Indigo Carmine, Indigotine is a direct dye similar in
color to Niagara Sky Blue 6B ; ^ gram in 95 ml. of distilled water,
5 ml. of 5 per cent sodium sulfate, and ^ ml. of acetic acid. The
stain gives an indigo on sections of animal tissues within ten min-
utes, on plant tissues in one to 24 hours. An important property
of the dye is its selectivity in staining nuclei, and its delicate shading
of embryonic or plant meristematic tissues. These minute differ-
ences in muscular, vascular, nervous, and digestive tissues are evi-
dent after a triple or quadruple stain including : ( 1 ) a mixture of
nine parts Sun Yellow and one part Acid Fuchsine for ten minutes
followed by Indigo Carmine for five to ten minutes; or (2) a mix-
ture of ten parts Sun Yellow, five parts Orange II, and one part
Acid Fuchsine, followed by Indigo Carmine.

Chapter VII
BIOLOGICAL STAINS

B. Natural Organic Dyestuffs

Since prehistoric times numerous natural dyes have been used in
the dyeing of fabrics. It was not until the discovery of the art of
mordanting, however, that any considerable advance in dyeing be-
came possible. The mordants at first employed consisted of the
naturally occuring sulfates of iron and aluminum.

1239. Carmine, Carminic Acid, or Cochineal is an acid mor-
dant dye, light 3 to 4. Cochineal is extracted with water contain-
ing ammonium alum from the tropical insect, Coccus cacti, reared
on cactus plants in Mexico. The purified red pigment is known
as Carmine. The active coloring principle of this dye is Carminic
Acid, which, upon the addition of ammonium alum, potash alum,
borax, sodium stannate, or sulfuric acid becomes soluble in water.

The stain is prepared either from the dry powdered cochineal
bugs or from the purified Carminic Acid.

1 . A saturated stain contains 0. 1 gram of Carminic Acid or the
extract from 0.5 gram of powdered bugs in 100 ml. of 0.2 per cent
sulfuric acid at 50° C. ; several hours is necessary to effect complete
solution. Filter the hot liquid and add 20 per cent alcohol by vol-
ume. Invertebrates or chick embryos are placed in a tin mordant
for 30 minutes ; they are stained a brilliant scarlet within 30 min-
utes at 50° C. This solution is unsatisfactory for paraffin sections
unless the material is first stained in bulk.

2. Dissolve 0.1 gram of Carminic Acid or extract 0.5 gram of
cochineal bugs in 100 ml. of 1 per cent ammonium acetate in 20 per
cent alcohol. A brilliant carmine is obtained on whole mounts of
invertebrates with a 30-minute mordant in 5 per cent ammonium
alum and a stain for 30 minutes at 50° C. This ammonium acetate
solution may be used for paraffin sections although the red devel-
oped in 24 hours is not so good as the color obtained by staining in
bulk.

3. Carmine dissolves in a solution of 5 per cent ammonium alum

66

STAIN TECHNIQUE 67

in 20 per cent alcohol. The alum stain yields lavender or fuchsia
with aluminum, tin, or copper mordants; blue-black to black with
uranium or iron salts. The alcohol added to the stain serves as a
preservative.

1242. Litmus, Archil, Orcein is a dye extracted from different
lichens. Its principal value is that of an indicator.

1243. Brazilin, Brazil Wood is an acid mordant dye, light 5.
The tropical redwood Caesalpinia, family Leguminosae, contains
Brazilin which is soluble in water or alcohol, and is changed on oxi-
dation to a red solution of Brazilein. Plant tissues are stained car-
mine to dull brown but the color is of low fastness.

1246. Haematoxylin, Hematein is an acid mordant dye, light
2 to 4. The dye is obtained from the wood of Haematoxylon cam-
pechianum, family Leguminosae, indigenous to Mexico and Central
America but cultivated in the West Indies. When freshly cut the
wood is colorless ; upon exposure to air, the surface of the wood be-
comes reddish brown with the development of Haematoxylin from
a glucoside. After chipping the wood, the dye is extracted with
boiling water in open pans or with steam under pressure in closed
vessels. The extract is concentrated to a liquor or a solid. On
further treatment, the crude product yields the light brown crystals
of Haematoxylin.

The fastness of Haematoxylin differs with (1) the various
metallic salts used as mordants, (2) the kind of staining solution,
and (3) the temperature of application. The color obtained with
iron Haematoxylin has a fastness of 3 to 4 ; of alum Haematoxylin,
3. By using iron alum as a mordant and the stain ripened in dilute
alcohol, the color developed at 50° C. has a fastness of 2, almost
the equal of the best coal-tar dyes.

Directions for making the solution as originally given by J.
Delafield in Zeit. Wiss. Mik.2: 288 (1885) are: "To make 600 cc
of the solution, take 400 cc saturated aqueous solution of ammonium
alum and add to this 4 grams of crystallized Haematoyxlin dis-
solved in 25 cc strong alcohol. This at first produces a light violet
or sometimes a dirty red color, but on exposure to the light in an
unstoppered bottle the color deepens and a light precipitate forms.

68 W. H. E M I G

After standing for three or four days exposed to the air and light,
the solution is filtered and 100 cc each of glycerin and methyl alcohol
are added. The solution is now allowed to stand until the color is
sufficiently dark and is then filtered and kept in a tightly stoppered
bottle. This solution must be considerably diluted with water be-
fore using." (N. Y., April 18th, 1885.)

This original formula of Delafield (as given above) contains a
number of errors. It is impossible to dissolve four grams of
Haematoxylin in 25 ml. of strong alcohol because the solubility of
the dye is approximately £ per cent. Therefore, if only the dis-
solved portion of the dye is added to the alum solution, about 95 per
cent of the crystals remain unused. Since Haematoxylin is slightly
more soluble in ammonium alum than in alcohol, it is unnecessary
to use alcohol as a solvent. As soon as alcohol is added to aqueous
ammonium alum, the few crystals first thrown out of solution serve
as centers for the rapid growth of larger crystals. Ammonium
alum is soluble in water but insoluble in alcohol. At the time the
stain is filtered, a considerable quantity of the dye and half of the
alum remain undissolved. Since it has long been the practice of
technicians to employ concentrated stains, a saturated solution of
Haematoxylin in ammonium alum, then, is evidently what Delafield
wished to obtain.

Taking into consideration the characteristic properties of
Haematoxylin as a dye, a saturated stain containing ammonium
alum is made in accordance with the formula given below.

Alum Haematoxylin (aluminum Haematoxylin).

Distilled water .... 160 ml., the solvent

Alcohol, 95% 40 ml., the preservative

Ammonium alum . . 10 grams, the mordant

Haematoxylin 1 gram, optimum solubility

This staining solution is ripened in three days if kept at 60° C. in a
liter Erlenmeyer flask plugged with cotton. After having been
ripened, the stain is filtered. By adding 20 ml. of glycerin, which
retards the oxidation of the stain, the keeping qualities are im-
proved. Should methyl alcohol instead of ethyl alcohol be added

STAIN TECHNIQUE 69

to the solution, the color produced by the stain is more reddish
violet.

Iron Haematoxylin is J per cent Haematoxylin in water or
dilute alcohol. The mordant, 1 to 4 per cent ferric ammonium sul-
fate, is applied separately. Heidenhain gives detailed directions for
the preparation and use of the stain (Martin Heidenhain, "Noch
einmal iiber die Hamatoxylinfarben," Zeit. Wiss. Mik. 13: 186-
199 (1896)). He reports the application of iron Haematoxylin
by Benda on nervous tissue, by Biitschli on cytoplasm, and by
Sobotta on tissues fixed in vapors of osmic acid. Heidenhain
recommends 2J to 4 per cent ferric ammonium sulfate, because
weaker solutions decompose quickly. Paraffin section on slides are
placed vertically in the mordant six to twelve hours. The stain is
given as Weigert's solution containing 1 gram of Haematoxylin in
10 per cent alcohol. Four weeks are needed for the ripening, and
then, the stain is diluted with an equal volume of distilled water.
Tissues are stained 24 to 36 hours, rinsed, differentiated in 2^ per
cent ferric alum, and washed in running water 10 to 15 minutes.

Iron Haematoxylin is applied in two solutions : the mordant
ferric ammonium sulfate and an aqueous stain.

A. The mordant.

Distilled water 100 ml.

Ferric ammonium sulfate, clear

lavender crystals 1 gram, or as much

as 4 grams

B. The stain.

Distilled water 160 ml.

Alcohol, 95% 40 ml., the preservative

Haematoxylin 1 gram

The stain is ripened, in a liter Erlenmeyer flask plugged with
cotton, at 60° C. for two days. The 1 per cent ferric alum should
not be kept more than three days because a trace of sulfuric acid,
which appears in dilute solutions of ferric alum on standing, pro-
duces a greenish Hematein pigment.

A definite correlation exists between the fixatives, tissues, and
stains. Using alum Haematoxylin, a tissue fixed in formalin

70 W. H. E M I G

presents a general stain while the material taken from chrom-acetic
yields a nuclear stain. Similar results are obtained with iron
Haematoxylin. Should sublimate-acetic be used as the fixative, the
results differ : a general stain is obtained whenever the material is
left in the dye only long enough to provide the color pigment; by
overstaining in Haematoxylin and destaining in iron alum, the re-
sult is a nuclear stain.

Staining with Haematoxylin. The procedure in which the re-
action is checked as soon as sufficient color has been produced is
known as the progressive method of staining. Alum Haematoxy-
lin is a progressive stain. The ripened solution stains animal
tissues within five minutes, plant tissues in 15 to 30 minutes.
Should the material be overstained in a few minutes, the solution
may be diluted with water.

The regressive method of staining is followed when the dye
accumulates to excess, whereupon the surplus pigment must be re-
moved later with a suitable solvent or reducing agent. Iron
Haematoxylin is a regressive stain. For a rapid schedule, animal
tissues are mordanted in 1 per cent ferric alum 15 minutes, rinsed,
stained in ripened Haematoxylin one minute, differentiated in 0.25
per cent ferric alum for five minutes, rinsed, left in 0.01 per cent
ammonium hydroxide three minutes, and rinsed with distilled
water. With a longer schedule, the material is left in 2 to 4 per
cent ferric alum 12 to 24 hours, rinsed, stained 24 hours, differen-
tiated in 1 per cent ferric alum, rinsed, left in 0.01 per cent am-
monium hydroxide three minutes, and rinsed with distilled water.
Plant tissues are mordanted in 1 per cent ferric alum 30 minutes,
rinsed, stained in Haematoxylin three minutes, differentiated in
0.25 per cent ferric alum five to ten minutes, rinsed, left in 0.01 per
cent ammonium hydroxide three minutes, and rinsed with .distilled
water. A slower schedule requires 24 hours in the mordant and 24
hours in the stain; the blue-black pigment is so fast in root tips
there is no evidence of fading after twenty years.

As a rule, a tissue prepared for cytological studies does not re-
quire a counterstain. Under low power objectives, a trace of color
in the cytoplasm aids in bringing out the third dimension of the

STAIN

TECHNIQUE 71

image. Naphthol Green B is well suited as a counterstain because
generally it is applied with iron alum, is fast to light, and does not
extract any of the Hematein. After the sections have been stained
in iron Haematoxylin and differentiated in iron alum, the material is
rinsed a few seconds in distilled water and counterstained in Naph-
thol Green B for five minutes.

1249a. Chlorophyll, Carotene (Carotin), and Xanthophyll are
naturally occurring pigments, light 4. In microscopic plants, the
green pigment may be fixed with the aid of copper acetate; this
fixation is probably a replacement of the magnesium in chlorophyll
by copper.

Fixative, methyl alcohol- formalin-acetic

Methyl alcohol 100 ml.

Formalin, 40% commercial 100 ml.

Acetic acid, 10% 100 ml.

Copper acetate 1 gram

Because chlorophyll is soluble in alcohol, fixed materials for perma-
nent mounts in their natural colors are washed in water and dehy-
drated from 10 per cent glycerin.

C. Inorganic Colouring Matters

1288. Prussian Blue, Berlin Blue, Chinese Blue is ferric fer-
rocyanide, light 1 to 2. The pigment may be used as a stain for
whole mounts of algae, fungi, or invertebrates. The material is
placed first in 3 per cent potassium ferrocyanide for 30 minutes,
rinsed thoroughly with distilled water, and then covered with a solu-
tion of 1 per cent ferric ammonium sulfate. Should the color be
too light, staining may be repeated until the desired shade is ob-
tained. The 10 per cent glycerin used for dehydration should con-
tain a few drops of J per cent potassium dichromate so that the
ferric ferrocyanide does not change to the soluble and colorless
ferrous ferrocyanide. Prussian Blue is stable in either balsam or
glycerin.

INDEX

Colour Index Numbers

Acetin Blue R, 860
Acid Alizarin Blue GR, 1048
Acid Alizarin Green G, 1049
Acid Azofuchsine, 30, 692
Acid Blue B, 736
Acid Blue G, 712
Acid Bordeaux, 88
Acid, Carminic, 1239
Acid Fuchsine, 692
Acid Green G, 670
Acid Magenta, 692
Acid Orange G, 27
Acid Orange II, 151
Acid, Picric, 7
Acid Rubine, 692
Acid Violet, 698
Acid Yellow, 16
Acridine Orange, 788
Acridine Red, 740
Acridine Yellow R, 2R, 785
Aldehyde Green, 676a
Alizarin, 1027
Alizarin Blue S, 1067
Alizarin Cyanine G, 1051
Alizarin Cyanine R, 1050
Alizarin Green G, 917
Alizarin Red S, 1034
Alizarin Sapphire BN, 1054
Alizarin Sodium Sulfonate, 1034
Alizarin Yellow G, 36
Alizarin Yellow R, 40
Alkali Blue 6B, 703
Alkali Green, 665
Alkali Green D, 594
Amaranth, 184

Aniline Blue, alcohol soluble, 689
Aniline Blue, water soluble, 705,
706, 707

Aniline Green, 676a
Aniline Red, 677
Aniline Yellow, 9
Anthracene Green, 783
Archil, 1242
Auramine, 655
Aurin, 724
Azo Blue, 463
Azo Bordeaux, 88
Azocarmine GX, 828
Azofuchsine, 30
Azorubine, 179
Azophor Red PN, 44
Azure A, B, C, 923
Azure I, 11,923
Basic Fuchsine, 677
Benzaldehyde Green, 676a
Benzoazurine G, 502
Benzopurpurin B, 450
Benzopurpurin 4B, 448
Benzopurpurin 10B, 495
Benzyl Violet, 683
Berlin Blue, 1288
Biebrich Scarlet, 280
Bismarck Brown G, Y, 331
Bismarck Brown R, 332
Blue Lumiere, 689
Bordeaux, 88
Brazil Wood, 1243
Brazilin, 1243
Brazilein, 1243
Brilliant Acid Blue A, 714
Brilliant Acid Blue V, 712
Brilliant Alizarin Blue, 931
Brilliant Blue, 877
Brilliant Cresyl Blue, 877
Brilliant Green, 662
Brilliant Scarlet 3R, 185

72

STAIN TECHNIQUE

73

Brilliant Yellow, 364
Capri Blue, 876
Carbanthrene Blue GD, 1113
Carbanthrene Jade Green, 1101
Carbanthrene Red BN, 1162
Carbanthrene Violet 2R, 1 104
Carotene, 1249a
Carmine, 1239
Carminic Acid, 1239
Carmoisine, 252
China Blue, 707
China Green, 657
Chinese Blue, 1288
Chlorazol Black E, 581
Chlorophyll, 1249a
Chromotrope 2R, 29
Chrysamine G, 410
Chrysoidine R, 21
Chrysoidine Y, 20
Chrysophenine, 365
Coccinine, 120
Cochineal, 1239
Cerulein MS, 783
Coerulein MS, 783
Congo Corinth G, 375
Congo Red, 370
Coralline, 724
Cotton Blue, 706, 707
Crocein Scarlet MOO, 252
Crocein Scarlet 5R, 280
Crocein Scarlet 7B, 286
Crystal Violet, 681
Cyanine, 806
Cyanosine, 776
Cyanosine B, 780
Dahlia, 679

Delafield's Haematoxylin, 1246
Diamine Blue 3B, 477
Diamond Fuchsine, 677
Diamond Green, 657
Diazine Green B, 593
Direct Black E, 581
Direct Fast Orange, 326

Direct Fast Scarlet 4BS, 327

Direct Green B, 593

Direct Green G, 594

Direct Sky Blue, 520

Eosin B, 771

Eosin Y, 768

Erie Black GXOO, 581

Erie Fast Orange, 326

Erie Fast Red FD, 419

Erie Fast Scarlet YA, 326

Erie Garnet B, 375

Erie Violet BW, 387

Erie Violet 3R, 394

Eriochrome Azurol V, 720

Erythrosin B, 773

Erythrosin G, Y, 772

Ethyl Eosin, 770

Ethyl Green, 662

Ethyl Violet, 682

Fast Green FCF, 670

Fast Red A, 176

Fast Red S, 16

Fluorescein, 766

Fuchsia, 842

Fuchsine S, 692

Gallein, 781

Gallocyanine, 883

Gentiana Blue 6B, 689

Gentian Violet, 681

Gentian Violet 6B, 683

Gold Orange, 151

Haematoxylin, 1246

Heidenhain's Haematoxylin, 1246

Helianthine, 142

Hematein, 1246

Hoffmann Violet, 679

Indigo, 1177

Indigo Carmine, 1180

Indigotine, 1180

Induline, alcohol soluble, 860

Induline, water soluble, 861

Iodine Green, 686

Iodine Violet, 679

74

W. H. E M I G

Iron Haematoxylin, 1246

Isamine Blue 6B, 710

Janus Green B, 133

Janus Red B, 266

Lanacyl Violet B, 207

Lanacyl Blue BB, 210

Light Green SF, 670

Litmus, 1242

Magdala Red, 857

Magenta, 677

Malachite Green, 657

Martius Yellow, 9

Mauvine, 846

Meldola Blue, 909

Metanil Yellow, 138

Methyl Blue, 705, 706

Methyl Eosin, 769

Methyl Green, 684

Methyl Orange, 142

Methyl Red, 211

Methyl Violet B, 2B, 680

Methyl Violet 5B, 6B, 683

Methyl Violet 10B, 681

Methylene Azure, 923

Methylene Blue, 922

Methylene Green, 924

Methylene Violet, 842

Mikado Yellow G, 622

Naphthol Green B, 5

Naphthol Yellow S, 10

Naphthyl Red, 856

Naphthylamine Brown, 175

Naphthylamine Red, 184

Naphthylamine Yellow, 9

Neutral Red, 825

Neutral Violet, 826

New Fuchsine, 678

New Magenta, 678

New Methylene Blue, N, NSS, 927

Niagara Sky Blue, 520

Niagara Sky Blue, 6B, 518

Night Blue, 731

Nigrosine, water soluble, 865

Nile Blue Sulfate, 913

Opal Blue, 689

Orange G, 27

Orange R, 161

Orange II, 151

Orange III, 142

Orange IV, 143

Orcein, 1242

Para Red, 44

Pararosaniline, 676

Patent Blue A, 714

Phenolphthalein, 764

Phloxine B, 774, 778

Phosphine, 793

Picric Acid, 7

Ponceau G, 2G, 28

Ponceau R, 2R, 79

Ponceau 3R, 80

Ponsol Golden Orange RRT, 1097

Ponsol Red 5GK, 1131

Pontachrome Brown MW, 101

Pontachrome Orange R, 415

Pontacyl Carmine 6B, 57

Pontacyl Carmine 2G, 31

Pontamine Fast Pink BL, 353

Pontamine Fast Scarlet 4BS, 327

Primuline, 812

Prussian Blue, 1288

Purpurin, 1037

Pyronine B, 741

Pyronine 2G, 739

Quinoline Yellow, 801

Red Violet 5R, 679

Rhodamine B, 749

Rhodamine G, 750

Rhodamine 6G, 752

Rhoduline Blue GO, 926

Rose Bengale, 777

Rose Bengale B, 779

Roseine, 677

Rosolic Acid, 724

Rubine S, 692

Safranine A, G, O, Y, 841

STAIN TECHNIQUE

75

Scarlet 2G, 28

Scarlet R, 74

Scarlet R, 2R, 79

Setoglaucine O, 658

Solid Green, 657

Soluble Blue, 705, 706, 707

Spirit Blue, 689

Stilbene Yellow, 622

Sudan II, 73

Sudan III, 248

Sudan IV, 258

Sulfindone Brilliant Blue CG, 957

Sulfogene Bordeaux BRN, 1012

Sulfogene Brilliant Blue 3GCF, 957

Sulfogene Brilliant Blue 6BS, 957

Sulfogene Brilliant Green 2G, 1006

Sulfogene Direct Blue BRS, 956

Sulfogene Green 2B, 1006

Sulfur Bordeaux, 1012

Sulfur Bordeaux BCF, 1012

Sulfur Brilliant Blue, 957

Sulfur Brown 4G, 949

Sulfur Direct Blue, 956

Sulfur Green, 1006

Sulfur Green 3G, 1006

Sulfur Orange, 948
Sulfur Yellow, 948
Sun Yellow, 620
Superchrome Black PV, 170
Superchrome Garnet Y, 168
Superchrome Violet B, 169
Tartrazine, 640
Thionine, 920
Thionine Blue GO, 926
Toluidine Blue O, 925
Toluylene Red, 825
Tolyl Blue 5R, 289
Trinitrophenol, 7
Tropaeolin O, 148
Tropaeolin OO, 143
Tropaeolin OOO, No. 2, 151
Trypan Blue, 477
Trypan Red, 438
Uranine, 766
Vesuvine R, 332
Victoria Blue B, 729
Water Blue, 707
Wool Black B, 315
Xanthophyll, 1249a