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^ Marine Biological laboratory Library

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Presented by |i

John Wiley and Sons, Inc.
July, 1959

o

PRINCIPLES OF
BIOLOGICAL MICROTECHNIQUE

PRINCIPLES OF
BIOLOGICAL MICROTECHNIQUE

A Study of Fixation and Dyeing

by
JOHN R. BAKER

D.Sc.(Oxon.), Reader in Cytology in the University of

Oxford; Joint Editor of the Quarterly Journal

of Microscopical Science

LONDON: METHUEN & CO LTD
NEW YORK: JOHN WILEY & SONS INC

First published in 1958

Catalogue No 600S ju
© 1958 by John R. Baker

Printed in Great Britain
by Richard Clay and Company Ltd, Bungay, Suffolk

DEDICATED
TO THE MEMORY OF

PAUL EHRLICH

GENIUS

IN CHEMOTHERAPY

IMMUNOLOGY

:\TICROTECHNIQUE

Prefc

ace \^

The principles of biological microtechnique may perhaps be re-
duced to one — the principle that when we make a microscopical
preparation of any sort, we ought to try to understand what we are
doing, for otherwise we shall examine an unknown object that has
been treated in an unknown way. A scientific outlook has been
introduced into certain branches of our subject, particularly histo-
chemistry, but there are others in which rule of thumb rules indeed.
One thinks at once of the most ordinary processes of the histo-
logical or cytological laboratory: of fixation, embedding, dyeing,
and mounting. Here the empirical outlook is often manifest, and
some workers are content to follow the recipe-book blindly, as
though a scientific result could be obtained by unscientific means.

A very long book — and a very learned author — ^would be neces-
sary if the attempt were made to illustrate the principles of micro-
technique by a full consideration of all its branches. It seems best
to concentrate on the most familiar processes, so that the principles
may find most frequent application in practice. In fixation and in
dyeing the tissues are responsive : they react to what we do to them.
In embedding and mounting they are more passive, allowing us
to surround them with what we w^ill. I therefore choose fixation
and dyeing as being even more interesting than the other familiar
branches of microtechnique. I hope that a study of the principles
that should guide us in these branches may engender an outlook
towards microtechnique that will find application in a wider field.
This outlook is at least as necessary when tissues are prepared for
the electron microscope as in our more homely endeavours within
the realm of light.

The book is addressed to research-workers, teachers, and
students in the fields of pathology, histology, cytology, zoology,
and botany. The primary intention has been to make it as useful
and attractive as possible to the consecutive reader, but certain
features have been introduced to help the casual inquirer. Thus
there are numerous cross-references and rather a lot of repetitions.
Three of the chapters (5, 6, and 9) can be used as though they were
parts of a work of reference. A full index is provided.

vii

VIU rKE-rA»^ii

This is in no sense a text-book. There is, I hope, nothing dog-
matic in it. Its purpose is as much to show the gaps in knowledge
as to knit together what is surely known. The possibilities for re-
search in microtechnique seem endless, and every effort has been
made to point out as many of them as possible.

The book contains a good deal of new material of three sorts.
First, there are new contributions to the theory of fixation and dye-
ing. Secondly, there are many factual observations that have not
been published previously. Thirdly, the Appendix contains full
descriptions of new experiments illustrating the principles under-
lying the processes of fixation and dyeing. Most of these can be
carried out in practical classes.

There is in the whole book no practical instruction on how to
make a microscopical preparation. For this the reader should turn
to one of the excellent guides to the subject, such as Langeron's
Precis de microscopie^'^^^ or Pantin's Notes on microscopical technique
for zoologists. ^^^

Apart from my own little book on Cytological technique, "^^ I know
of only three that cover more or less the same field as the present
work. These are Fischer's Fixierung, Fdrbung und Ban des
Protoplasmas (1899),^^^ Mann's Physiological Histology (1902),^"^
and Zeiger's Physikochemische Grundlageft der histologischen
Methodik (1938).^^^ There cannot be many equally important
fields of science to which so few books have been devoted — and
those few of such merit as these three.

In the historical parts of the book I have adopted the usual con-
vention of giving as the date of a discovery the year in which it was
first made known in print.

Acknowledgements. It was Mr Frank Sherlock, Head Technician
of the Department of Zoology, Oxford, w ho first gave me instruc-
tion in microtechnique, and I have always owed a debt of gratitude
to him. Dr H. M. Carleton was generous with good advice through-
out our association of thirty-five years. Prof. A. C. Hardy, F.R.S.,
has encouraged cytological studies in his Department and I am
thankful for all that he has done to help me. Dr M. Wolman
generously sent me the proof of his article on fixation^^^ before it
was published. One learns best, perhaps, by continual association
with lively young minds, and it has been my good fortune for many
years to have a splendid succession of research-pupils from many
lands, to whom I owe much. I have alwa3^s been lucky in my assist-
ants and must particularly mention Mrs B. M. Jordan-Luke,

PREFACE IX

whose skill in microtechnique has been of great benefit in my re-
search. She has given much practical help in connexion with the
experiments described in the Appendix. Mrs J. A. Spokes has
helped me with this book more than anyone else, by acting as my
secretary with uniform accuracy and good nature. Several of the
illustrations have been copied photographically from old books and
journals. ]\Ir P. L. Small and Mr J. S. Haywood have taken a lot
of trouble to produce as good copies as possible. Dr W. G. B.
Casselman has most kindly made several readings of the oxidation-
potentials of fixatives specially for this book.

I take the opportunity of mentioning the benefit I have derived
during the last eleven years from my association with Dr C. F. A.
Pantin, F.R.S., in joint editorship of the Quarterly Jour?ial of
Microscopical Science.

John R. Baker
Cytological Laboratory,

Department of Zoology,
University Museum,
Oxford.

Contents

Preface page vii

List of Illustrations xiii

PART i: FIXATION

1 Introduction to Fixation 19

2 The Reactions of Fixatives with Proteins, i. The Visible

Effects 31

3 The Reactions of Fixatives with Proteins. 2. The Chemi-

cal Changes 44

4 The Reactions of Fixatives with Tissues and Cells:

Methods of Research 66

5 Primary Fixatives Considered Separately, i . Coagulants 89

6 Primary Fixatives Considered Separately. 2. Non-

coagulants III

7 Fixative Mixtures 139

PART II: DYEING

8 Introduction to the Chemical Composition of Dyes 155

9 The Classification of Dyes 169

10 The Direct Attachment of Dyes to Tissues 187

1 1 The Indirect Attachment of Dyes to Tissues 207

12 The Differential Action of Dyes 228

13 Metachromasy 243

14 The Blood Dyes 262

15 Introduction to Vital Colouring 274

16 The Mode of Action of Vital Dyes 284

17 A Comparison between Dyeing and other Processes of

Colouring 296

xi

APPENDIX

1 The composition of solutions expressed as percentages :

conventions adopted in this book page 313

2 Experiments on fixation 314

3 Experiments on dyeing 321

4 Use of the word 'chromatin' 327

5 Notes on speUing 329

List of References 331

Index 345

Illustrations

Fig. I . Graphical representation of the changes in volume
undergone by gelatine /albumin gels during 1 8 hours
in various fixatives page 36

Fig. 2. Pipettes used in the measurement of the rate of

penetration of fixatives into gelatine /albumin gel 38

Fig. 3 . Graph showing the rate of penetration of fixatives

into gelatine /albumin gel 39

Fig. 4. Protein coagula seen under the microscope 41

Fig. 5 (plate), a, lobes of the liver of the rabbit left for 25
hours in fixatives and then cut across

B-E, photomicrographs illustrating Young's ex-
periments on the addition of indifferent salts to
fixatives faci?tg page 67

Fig. 6. A cell from the intestine of Oniscus (woodlouse),
fixed in mercuric chloride : to show the coagulation
of protoplasm page 67

Fig. 7. Graph showing the thickness of rabbit-liver fixed
by a saturated aqueous solution of mercuric chlor-
ide in various times 68

Fig. 8 (plate). The effect of fixatives on cultured cells from
the chorioid or sclerotic coat of the eve of the chick
embyro facing page 70

Fig. 9 (plate). Sections of the testis of the mouse, to show

good and bad fixation 74

Fig. 10. Outlines of the fully-grown primary spermatocyte
of the snail, Helix aspersa, to show the effect of fixa-
tion and subsequent treatment on the size of the
cell page 79

Fig. 1 1 . Graph showing the effect of fixation and subsequent
treatment on the volume of the nuclei of cartilage-
cells 80

xiii

XJV ILLUSTRATIONS

Fig. 12. Graph showing how the volume of the eggs of Ar-
bacia pustulosa is affected by the addition of non-
fixative sahs to formaldehyde solution P^S^ 82

Fig. 13. Diagram showing the coefficient of elasticity of the

belly-muscle of the cat, fixed in various ways 87

Fig. 14. Graphical representation of the ions present in a
2*5% aqueous solution of potassium dichromate
and in a solution of chromium trioxide containing
the same weight of chromium 105

Fig. 15. Photomicrographs of lecithin smeared on glass. A,
in distilled water, showing outgrowth of myelin
forms; B, in a concentrated solution of calcium
chloride, showing absence of myelin forms 115

Fig. 16. Three Ringkorner and a cap or hood (Kapuze)
formed by partial solution of lipid globules : osmium
preparations 125

Fig, 17. Graph showing the transmission of light through a
layer i cm thick of basic fuchsine, 0-00062%
aqueous 161

Fig. 18. Graph showing the reciprocals of the transmission
of light through a layer i cm thick of basic fuchsine,
0-00062% aqueous 162

Fig. 19. Graph showing the optical density of a layer i cm

thick of basic fuchsine, 0-00062% aqueous 163

Fig. 20. Graph showing the transmission of light through
a layer i cm thick of acid fuchsine, 0-00293%
aqueous 165

Fig. 21 (plate). Haematoxylon campechianum facing page 172

Fig. 22 (plate). The cochineal insect and its food-plant 176

Fig. 23 (plate). Apparatus for cataphoretic experiments

with dyes 189

Fig. 24 (plate). Ehrlich at the age of 24 193

Fig. 25. Diagrammatic representation of the dyeing of
collodion by typical basic, amphoteric, and acid
dyes page 194

Fig. 26. Diagrammatic representation of the dyeing of gela-
tine by typical basic, amphoteric, and acid dyes 1 95

ILLUSTRATIONS

XV

Fig. 27. Graph showing the transmission of hght of various

wave-lengths through toluidine blue solution page 250

Fig. 28 (coloured plate), a, human blood from a patient
with myeloid leucaemia; coloured by Ehrlich's
'Triacid' dye (from Ehrlich & Lazarus^^^)

B, normal human blood dyed by Leishman's
method (from Carleton & Short, ^^^ by permission
of Messrs Longmans, Green & Co.) facing page 264

Fig. 29 (plate). Ehrlich at about the time when his work on

vital dyes was merging into chemotherapy 274

PART ONE

FIXATION

B

CHAPTER I

Introduction to Fixation

Every cytological investigation should start, if possible, with the
study of the living cell. A lot of useless controversy would have
been avoided if this guiding principle had been obeyed, for too
much reliance has been placed on the study of dead cells. It may
be queried, then, why we do not restrict ourselves to the study of
living material. There are four main reasons.

(i) When tissues are cut in thin sections, it is very easy to
determine the relations of the cells to one another and to the inter-
cellular material ; the structure of the cells themselves is often very
clearly revealed. Some kinds of cells cannot be isolated for vital
study, and these are best examined in sections. There are usually
practical difficulties in cutting sections of living tissues thin and
uniform enough for convenient microscopical study, and anyhow
living cells are necessarily damaged by being divided. It is easy to
cut thin, uniform sections of dead tissues that have been treated
in particular ways.

(2) Although vital colouring gives very important information,
yet it is not a method of general application, for many tissue-con-
stituents are not revealed by it. Almost all the constituents of dead
tissues can be dyed in brilliant, contrasting colours.

(3) Few histochemical tests are applicable to living cells.

(4) It is convenient to have permanent preparations.

We need an understanding of the kinds of changes that proto-
plasm undergoes when the processes of microtechnique are
applied to it: armed with that, we can profit greatly from the study
of dead material. Such understanding presupposes familiarity
with living material. In this book we are not directly concerned
with the techniques used in the study of living material, except
those of vital colouring, but a general knowledge of the structure
of living cells will be assumed throughout. We shall consider what
happens to the living cell when it is fixed, and to the fixed cell
when it is dyed.

19

20 FIXATION

It is necessary at the outset to distinguish between preservation
and fixation.

A small piece of tissue that has been cut out of an organism will
generally cease to retain its structure at the microscopical level
quite soon, unless special precautions are taken to keep the cells
alive. The main potential causes of damage are evaporation,
osmotic sw^elling or shrinkage, attack by bacteria or moulds, and
autolysis.

Autolysis is the self-digestion of cells by enzymes that are
always present wdthin them and presumably synthesize proto-
plasmic proteins during life. On the cessation of normal vital
activity their action is reversed, at any rate in the sum-total of its
effect. These enzymes are known collectively as kathepsin. Two
of them are proteinases, similar to pepsin and trypsin: these shorten
the protein chains to peptides. Two others are an aminopeptidase
and a carboxypeptidase, capable of pulling the terminal amino-
acids off the newly-exposed ends of the peptide molecules. As a
result there is a general dissolution of the proteins, which are
rendered no longer capable of coagulation by heat or chem-
ical agents. The rate of autolysis can be measured by finding
what proportion of the protein has become non-coagulable in

a given time. The process is slow at 6° C but still occurs even
ato°.i«2

To preserve a piece of tissue, then, it is necessary to place it
in a fluid that will neither shrink nor swell it, nor dissolve or
distort its constituent parts; will kill bacteria and moulds; and
will render kathepsin inactive. A fluid that will do this is a pre-
servative.

It would be very convenient to have fluids in which separate
cells or teased fragments of tissues might be indefinitely pre-
served and in which they could be examined microscopically at any
time. Although it has been customary for centuries to preserve
organisms and their parts in suitable fluids for subsequent study
with the naked eye or hand-lens, not many cytologists or histo-
logists have sought to elaborate fluids of this kind that would serve
their needs. The best-known of the few such fluids that exist are
Petit's (usually know^n as the fluid of Ripart and Petit ^^^) and
Amann's,^' '^ especially the latter's lactophenol. Amann called his
fluids Beohachtiingsmedien, to indicate that they not only prevented
the decay of tissues, but were media in which cells might remain
during microscopical examination. It is desirable that research

INTRODUCTION TO FIXATION 21

should be undertaken to find new, improved preservatives, but the
subject does not fall within the scope of this book.

A fixative must do everything that a preservative does, and
something else as well. We do not say that we fix a door open
when we merely open it: fixing implies that we take action to
ensure that it will retain its position when other forces subse-
quently act upon it. Similarly, the essence of fixation is that the
various tissue-constituents are modified in such a way that they
retain their form as nearly as possible when the tissue is subjected
to treatment that would have damaged them in their initial state.
It follows that fixation is a forward-looking process: it exists only
in relation to subsequent events.

The subsequent event in which the early microscopists were
chiefly interested was the cutting of sections with a hand-razor.
They wanted above all to make the tissues hard, and they called
the process 'hardening'. Some of their hardening fluids are used
as fixatives to the present day. When it was discovered that the
necessary support could be given to the tissues by embedding
them in collodion or other media, less emphasis was placed on
hardening, and the term 'fixation' came into general use in the early
eighteen-eighties.^^^

In modern microtechnique the processes against which it is
especially important that tissues should be protected are em-
bedding, sectioning, and mounting. The first- and last-named
often involve dehydration, which has a strong tendency towards
distortion; embedding often requires a high temperature; section-
ing can cause mechanical damage (especially cracking and
crumbling). A fixative is a fluid that stabilizes the tissue-constitu-
ents as far as possible against these and other potentially damaging
processes.

Although this is the primary function of fixatives, yet there are
others, scarcely less important. In particular, most fixatives make
the tissues much more easily colourable by dyes than when they
were alive, and colourable in particularly informative ways. After
suitable fixation almost every part of the cell and of the inter-
cellular material can be dyed, often with great selectivity, so that
neighbouring parts show up brilliantly in different colours.
Chromatin, which is scarcely colourable during life, is one of the
most easily dyed of all tissue-constituents after fixation. The
wealth of our knowledge of cytogenetics is to a large extent due to
this fact.

22 FIXATION

Living cytoplasm commonly has a refractive index (r.i.) in the
neighbourhood of 1-353;*^^'*^^ that is to say, not very much
higher than that of the saline solutions in which cells are commonly
immersed for vital study. An aqueous solution of sodium chloride
at 0*9% has an r.i. of i*335.^^-"- When cells are examined alive in
such media, a w^ater-immersion objective w^ill give almost as good
resolution as a first-rate oil-immersion objective, for the high
numerical aperture of the latter will be partly wasted. One may
surround living cells with innocuous media of the same r.i. as the
cytoplasm ** and thus obtain slightly higher resolution (as well as
gaining other advantages), but the difference will not be great. As
soon, however, as a fixative acts, a profound change occurs. The
evidence suggests ** that the protoplasm is now represented by
interlacing sub-microscopic fibres having the r.i. of dry protein
(about 1*54). These fibres lie in water, if the fixative is aqueous;
this can be replaced by media of any desired refractive index. If a
medium of r.i. close to that of dry protein is used (Canada balsam,
for instance), two results ensue: almost perfect transparency is
obtained (which may be modified as desired by the use of dyes),
and oil-immersion objectives can be used at their full aperture.
This fact should not, however, be too strongly stressed, for the
making of a permanent microscopical preparation involves con-
siderable shrinkage of the tissues of organisms (p. 76), and this
reduces or nullifies the advantage of higher microscopical resolu-
tion. The advantage can be fully secured only if the object to be
examined happens to be unshrinkable by the processes involved
in making a permanent preparation. The valves of diatoms provide
an example.

Some of the constituent parts of organisms do not require
fixation, because they are not subject to autolysis and are resistant
to bacteria and moulds and to most of the reagents ordinarily used
in microtechnique. Examples are chitin, cellulose, scleroproteins,
certain inorganic crystals, and amorphous silica. Most such sub-
stances not only do not need fixation but are not acted upon by
fixatives; or, if acted upon, may be dissolved (for instance, spicules
of calcium carbonate by fixatives containing acid). Many fixatives
leave droplets of triglyceride untouched ; these do not require to
be fixed if no lipid-solvent will be used subsequently.

Apart from such substances as these, it is the main purpose of
fixation to alter the tissue-constituents in such a way as to render
them no longer subject to autolysis, to decay through the action

INTRODUCTION TO FIXATION 23

of bacteria or moulds, or to distortion by subsequent treatment.
The framework of the cell is of protein, and disintegration would
occur instantaneously if this constituent were to disappear. Neither
lipid nor carbohydrate is essential for the cohesion of protoplasm.
For this reason it is always necessary to fix protein, whether other
substances are stabilized by a particular fixative or not. Fixation is
therefore primarily the stabilization of protein.

Fixation can be achieved either by chemical means or by the
application of heat. The latter method involves the coagulation of
proteins. It tends to cause distortion and does not commend itself,
as a general rule, in purely morphological studies. It was especially
recommended by Ehrlich, however, for the fixation of blood-
smears. He advised short treatment (J to 2 min.) at 110° C.^^^ In
a blood-smear the shape of the cells is anyhow distorted, but the
cell-contents may react with special clarity to dyes after heat-
fixation. The method could probably be used with advantage in
many histochemical studies. Its possibilities have been somewhat
overlooked.

Fixation by the use of reactive substances may be called
chemical fixation for short, without prejudgement of the question
whether chemical fixatives necessarily participate in all cases in
chemical reactions with tissue-constituents. Most of the sub-
stances used for fixation are solids, used in aqueous solution ; some
are liquids that can be used without the addition of water.

The number of substances that are really useful in chemical
fixation is very small. The substances may be divided into two
major groups, according to their effects on proteins. The members
of the two groups can easily be distinguished by testing their effect
on a solution of albumin (p. 32). Some of them act, like heat, by
coagulating the protein; others do not. The coagulant fixatives
usually transform protoplasm into a network, while the non-
coagulant do not. The principal fixative substances are listed on
p. 24. The ones not marked 'absolute' are used in aqueous solution.
(For the method of expressing percentage concentrations, see
Appendk, p. 313.)

The names used in this list are those of the undissolved sub-
stances. These names will be used throughout this book. Thus,
fixation by mercuric chloride or potassium dichromate must be
understood to mean fixation by some or all of the various ions that
are produced when mercuric chloride or potassium dichromate is
placed in water (pp. 99 and 126). This usage will be adopted for

24 FIXATION

two reasons. First, for simplicity: it would be tedious to have to
mention all the products of solution every time. Secondly, for
accuracy in the statement of concentrations. If, for instance, one
adds distilled water to 0-5 g of chromium trioxide to make 100 ml
of solution, the latter should not be called a 0-5% solution of
'chromic acid'; for the oxide takes up water in ionizing, and
the acid produced (cations plus anions) is present at more than

0-5 %•

Suitable

concentrations

for fixation

Coagulant
methanol
ethanol
acetone

100% (absolute)
100% (absolute)
100% (absolute)

nitric acid

0-5N

hydrochloric acid

0-5N

trichloracetic acid
picric acid
mercuric chloride

2% w/v

Saturated

Saturated

chloroplatinic acid

o-75% w/v

chromium trioxide

o-5% w/v

Non-coagulant

formaldehyde

4% w/v

osmium tetroxide .

I % w/v

potassium dichromate

1-5% w/v

acetic acid ....

5% v/v

In the experiments described in the succeeding chapters, the
substances were always used at the concentrations given in the
list, except where the contrary is distinctly stated. It will therefore
be possible to omit mention of concentration and thus avoid
unnecessary repetition.

Non-fixative or 'indifferent' substances, such as sodium
chloride or sodium sulphate, are often added to solutions of these
fixatives. The effects of this will be considered in a later chapter
(p. 80).

None of the listed substances has all the qualities of a perfect
fixative, and mixtures are therefore made with the intention of
combining the virtues of the ingredients. Most mixtures contain
two or more of the listed substances, and often an indifferent
substance as well. The principles that should guide the design of
fixative mixtures will be considered in chapter 7 (p. 139).

Mixtures are generally called by the names of those who intro-

INTRODUCTION TO FIXATION 25

duced them. It is usual to use the surname alone: thus 'Bouin'
means Bouin's fluid.

Chemical fixation is generally achieved by putting an organism
or part of it in a fluid. In micro-anatomy and gross histology it is
sometimes necessary to fix a large volume of tissue in a single
piece. The fixative may be injected into a blood-vessel and thereby
reach all depths in the piece at almost exactly the same mo-
ment. This results in an evenness of fixation that cannot be
achieved in any other way with large pieces. The total volume of
fixative that can be held in the blood-vessels is, however, small in
comparison with that of the tissue; the fluid that has been injected
has to diflPuse through their walls before it can exert any effect on
cells other than those of the blood-vessels; and many cells lie a
long way (on the microscopical scale) from any blood-vessel, even
a capillary. Perfusion should therefore not be used as a general
rule. In cytology it is seldom necessary or desirable. The single
cell that it is desired to study can easily be obtained by cutting out
a piece of tissue a millimetre or so in diameter. If this be simply
thrown into a much larger volume of fixative, the latter will reach
all parts without much delay. The rate of penetration of fixatives is
discussed in detail below (pp. 37, 67, 150).

Vapours are occasionally used instead of fluids, but most fixa-
tives are not volatile, and the method is applicable only to very
minute pieces. The advantage gained is that nothing is dissolved
out of the tissue.

It is possible to fix in two stages, by taking preliminary action to
stop autolysis and then applying a fixative fluid. Autolysis may be
stopped almost instantaneously by placing tissues in some harm-
less, non-fixative fiuid maintained at a very low temperature.
Isopentane chilled to below —160° C is suitable. Ice-crystals form
in the tissues, but if the temperature is low enough they are very
small, and surprisingly little damage is done. In the process of
'freezing- thawing', the tissue that has been chilled is simply
allowed to thaw in an ordinary fixative at room-temperature or
thereabouts.*^^ In 'freezing-substitution' an organic fixative fluid
replaces the ice directly, without any melting of the ice-crystals.
Ethanol *^^ or methanol ^^^ may be used. The alcohol is chilled to
— 40° C or lower before the tissue is transferred to it from the iso-
pentane, and then allowed gradually to warm up. It acts on the
protein in the total absence of fluid water. These methods have
not been sufliciently used to allow their value to be judged with

26 FIXATION

confidence, but high claims have been made for the faithful
stabilization of cellular structure by f reezing- substitution. ^^^ The
necessity to use a non-aqueous fixative limits choice severely.

A piece of tissue may be suddenly chilled and the ice completely
evaporated off from it while the low temperature is maintained.
This process of 'freezing-drying' is an old one.^ It is often
carried out in a very elaborate way, but quite simple apparatus is
nowadays available.^^^ After warming to room-temperature the
dry tissue may be fixed as desired. The main advantage of the
method, however, is that the tissue can be obtained in an unfixed
but fairly stable state, the stability being due to the absence of
water. This state is suitable for certain chemical and enzymological
studies. The subject does not fall within the scope of this book,
because neither fixation nor dyeing is directly involved.

Although it is the purpose of fixatives, as a general rule, to leave
the structure of the tissue so far as possible unchanged, yet this
cannot apply to its chemical composition. Indeed, it may be said
that the whole purpose of fixation is to alter the chemical com-
position of certain tissue-constituents. If the chemical composition
of the tissue were unaffected, the fixative would be without effect:
the cells, in fact, would be still alive or like recently dead cells.

It might be thought that the need to alter chemical composition
would render fixation inapplicable in histochemical studies, but
this is far from being true. Indeed, the service of fixation to histo-
chemistry is as important as its service to dyeing. Tests that would
smash a living cell are applicable when it has been fixed. Many
fixatives affect the proteins only, leaving DNA, RNA, lipids,
carbohydrates, and various inorganic constituents, unchanged;
and the proteins themselves, though radically altered, generally
retain reactive groups that respond to histochemical tests. The
retention of the original structure in the fixed tissue is nearly as
important in histochemical as in purely morphological studies,
for the purpose of histochemistry is to disclose the distribution of
particular substances in -^pace.

Although it is ■ '-Oi \ation to stabilize the form of

tissue-constituents ays desirable to preserve all of

them. In studies c . .lOoi s, lOr instance, it is generally best

to use a fixative i ...i will either destroy mitochondria or allow
them to be destroyed by subsequent treatment, for otherwise they
will obscure the view.

With such exceptions as this it is the purpose of fixatives to

INTRODUCTION TO FIXATION 27

make the structural resemblance between the final preparation and
the living tissue as close as possible.

The appearance given in a fixed microscopical preparation is
nearly always to some extent artificial. The artifacts (p. 329) are of
two main kinds: intrinsic, or caused by distortion of the original
tissue-constituents; and extrinsic, or caused by the deposition
within the tissue of extraneous objects (such as the mercury pre-
cipitate mentioned on p. 103). Intrinsic artifacts are of two kinds:
primary, or produced by the fixative itself and therefore visible
while the tissue still lies in the fixative ; and secondary, or produced
by subsequent treatment.

Intrinsic artifacts are much more frequent than extrinsic. On
the w^hole they have a more serious effect on cells than on inter-
cellular matter, though connective tissue fibres are often wrinkled
and intercellular spaces distorted. Cells are subject to several
different kinds of artificial changes.*^* Objects that are present in
the living cell may simply disappear. Lipid globules, for instance,
may be dissolved away. Protoplasm may be coagulated in the form
of networks or lumps. So frequent is the network appearance in
microscopical preparations that it was formerly supposed to be a
character of the living substance; but Hardy ^^^ showed in 1899
that fixatives produced just such a microscopical network when
they coagulated a solution of albumin, and that some fixatives
made a coarse and others a fine net. Another frequent artifact is the
thickening of membranes. Protein is often coagulated on the inner
side of the nuclear and cell-membranes. Retractions also occur.
The nuclear membrane is frequently pulled away from the sur-
rounding cytoplasm, and both this and the cell-membrane are
frequently wrinkled. Blebs of cytoplasm are often extruded from
the surfaces of cells directly exposed to the fixative. The technical
term for this process is clasmatosis, but it is often called bubbling.
The latter is a misleading term, for no gas is formed.

Only by reference to the living cell can the quality of a fixative
be judged. This fact can scarcely be too strongly emphasized. If,
however, a particular fixative gives faithful stabilization of struc-
ture with certain chosen cells that have been carefully studied in
the living state, we have reason to trust it with others that cannot
be isolated for vital study.

Fixation for electron microscopy presents us with special
problems. Electron micrographs that show objects of microscopic

28 FIXATION

size enable us to judge the quality of fixation at the level of resolu-
tion of the light-microscope. Thus, an object of microscopic size,
known to be smooth in outline in life, may appear irregular in an
electron micrograph, and the irregularities may be of such a size
that they would have been visible with the light-microscope in
life, had they existed. We have here clear evidence of faulty fixa-
tion for electron microscopy; and if the fixation is faulty at the
microscopical level, we cannot trust it at the submicroscopical.
So far as submicroscopical objects are concerned, we have no
direct means of knowing whether a fixative is reliable or not. It is
customary to rely upon a fixative that is known to give faithful
stabilization of form at the microscopical level. It would be safest
not to rely on the submicroscopical detail shown in electron
micrographs unless similar pictures are given after fixation by
several different fixatives, all known to be reliable at the micro-
scopical level.

Many cytologists have examined separate living cells with the
light-microscope and noticed the changes produced in them when
a fixative was run below the coverslip. The classical work of this
kind was done by Strangeways and Canti (1927) with cultured
cells, by dark-ground microscopy. The invention of the phase-
contrast microscope led to a whole series of similar studies. Such
work is of great interest and value, but it is of course an incom-
plete way of studying fixation, for only the primary artifacts are
usually studied, and no evidence is obtained about the ability of the
fixative to stabilize the form of the cellular components against
subsequent treatment. In fact, fixatives are not tested as such in
these studies, but only as preservatives.

A defect of the method described in the last paragraph is that
the cells studied are immediately exposed to the action of the
fixative. Now when a piece of tissue is fixed in the ordinary way,
the cells that are thus exposed and not protected by any special
membrane (such as the free border of the intestinal epithelium of
vertebrates) are often seriously distorted, while those below,
protected as they are by overlying cells or membranes, are well
fixed. Even in a piece of tissue only a millimetre or so in diameter,
the great majority of the cells are not superficial. It therefore
follows that one might reject a fixative wrongly on the basis of
observations made on separate cells.

It is realistic to test fixatives on small pieces of tissue that will be
embedded and sectioned, with subsequent dyeing and mounting

INTRODUCTION TO FIXATION 29

of the sections. Care should be taken to chose deHcate tissues as
test- objects, for some are so robust that they withstand the action
of indifferent fixatives. It would be proper to take several bits of
material fixed in each of the fluids that it is desired to compare,
and embed them in a variety of different media ; for a fixative that
gives good results with one embedding medium may fail badly with
another. A full investigation of this sort has never been undertaken,
but details of a test involving paraffin embedding will be given on
a later page (p. 72). Paraffin is probably the most drastic medium.

The minimum length of time during which a fixative should act
depends partly on its rate of penetration (see pp. 37, 67) and partly
on its mode of action. In general, the coagulant fixatives have
achieved their full effect at any particular depth in the tissue as soon
as they have penetrated to that depth at the concentration necessary
to cause coagulation; but some fixatives, especially formaldehyde
(p. 60), may continue to act progressively on the tissues after the
latter has been fully permeated. Unless a fixative produces an ex-
trinsic artifact, prolonged action is seldom harmful. Tissues may
be left at least three years in Bouin's fluid without harm.^^ It is
convenient as a general rule to fix overnight (about 18 hours).
Very much shorter periods are sufficient for the tiny pieces, only
about a millimetre thick, that are often used in cytological work;
but overnight fixation ordinarily causes no damage. If the proteins
of the interior of a piece of tissue have not been fully acted upon in
18 hours, it is unlikely that satisfactory fixation will be achieved
by prolonging the period. For this reason it is not sensible to try to
fix pieces of tissue several centimetres thick.

Careful instructions are often given as to the exact length of
time during which particular fixatives should act, but these may
often be disregarded. The truth of this may be established by
getting someone else to fix three similar bits of tissue in the same
fixative for 8, 16, and 32 hours. When these have been sectioned,
dyed, and mounted, it will be found difficult or impossible to
guess which is which, unless there happens to have been pro-
gressive deposition of an extrinsic artifact and this has not been
removed.

It is sometimes desirable to 'postchrome' or 'postosmicate' a
piece of tissue ; that is to say, to leave it for a long time in a solution
of potassium dichromate or osmium tetroxide after initial fixation.
(See pp. 129 and 126.)

30 FIXATION

After fixatives have acted, they must be washed out of the tissues
so as to prevent the formation of extrinsic artifacts by their incom-
patibiHty with fluids in which the tissue must subsequently be
placed. In some cases the fluids that must be used for other pur-
poses (for instance, the alcohols used for dehydration) themselves
act as solvents for a fixative; in other cases special methods of
washing out must be adopted. Since the methods of elimination
are specific to each fixative, they will be considered separately
in chapters 5 and 6.

When a tissue has been fixed, one may sometimes wish to pre-
serve it indefinitely before embedding. This applies especially on
scientific expeditions, when there are usually no facilities for
embedding. The fixative itself may be unsuitable for indefinite
preservation, either because it is volatile or because it will eventu-
ally produce artifacts. In such cases a post-fixation preservative
may be used. Solutions of ^-hydroxybenzoic acid and its esters
are suitable. ^^^ These fluids are of an entirely different nature from
the preservatives mentioned at the beginning of this chapter, for
they are quite unsuited to the preservation of fresh tissues.

CHA PTER

The Reactions of Fixatives with
Proteins 1. The Visible Effects

For reasons that have already been mentioned (p. 23), fixation
is primarily the stabilization of proteins. It will be best to study
the reactions between fixatives and proteins before turning to
the more complex events involved in the fixation of tissues and
cells.

Some of the long chains of amino-acids that make up the pro-
teins of plants and animals are wound into submicroscopic balls,
others are extended in long fibres, often interlacing with one
another and held together by chemical bonds of various kinds ^^^
to form elaborate networks in three dimensions. The proportion of
globular to fibrous proteins varies widely in different tissues, the
globular existing alone in certain body-fluids, the fibrous pre-
dominating in certain elements of the connective tissues of animals.
In the present state of knowledge it is not possible to assess the
relative amounts of each in protoplasm (cytoplasm and nuclear
sap), but it is supposed that the fibrous here predominate and
form a 'cytoskeleton' in the form of a submicroscopic net-
work.^^i

When fixatives are added to protein sols, there is usually (though
not always) a change in visible appearance. Sometimes there is an
aggregation of protein molecules into microscopic granules, which
are seen as a cloudiness in the fluid. Stronger action produces a
flocculus of visible particles, which may remain suspended or
fall gradually as a precipitate. Stronger action again may convert
the whole of the protein into a single, coherent clot. The appear-
ance of new interfaces between solid protein and surrounding
water results in the scattering of light and thus the production of
whiteness or colour: transparency is in some degree lost. Cloudi-
ness, flocculation, and clot-formation are grades or stages in a
single process of coagulation. The more concentrated the protein

31

32 FIXATION

sol and the more vigorous the fixative, the greater will be the tend-
ency of the coagulated particles to cohere in a single clot.

Some fixatives act in quite a different way. Instead of coagulat-
ing, they make protein sols more viscous or convert them into
gels ; if the protein is already a gel, they stiffen and stabilize it. In
these processes there is no dissociation of protein from water and
therefore no production of surfaces that will scatter light. Instead
of a coagulum there is a viscous sol or an aqueous gel, transparent
unless opaque matter has been deposited from the fixative itself.

To observe the effects of fixatives on proteins, it is convenient
to begin with naked-eye observations on what happens when they
are added to globular proteins in the form of aqueous sols. Experi-
ments of this kind were made more than half a century ago by
Fischer ^^^ and Mann.^^^ A convenient way of carrying out such
experiments is described in the Appendix (p. 315). The protein
used is egg-albumin.

For the benefit of those who may read this chapter without
having read the first, it is necessary to say once more that in all the
experiments with fixatives described in this book, the substances
were used at the concentrations shown on p. 24, except where the
contrary is distinctly stated.

When a coagulant fixative is added to an albumin sol, a coherent
coagulum usually appears at once and occupies most of the space
filled by the fluid; in some cases (picric acid, mercuric chloride,
chromium trioxide) it has fallen somewhat towards the bottom of
the tube by the next day. It is either white (ethanol, acetone, tri-
chloracetic acid, mercuric chloride) or coloured (picric and chloro-
platinic acids, yellow; chromium trioxide, orange). With methanol
and nitric acid it is at first in the form of a fine, white flocculus ; the
particles eventually cohere, and indeed nitric acid produces in the
end rather a firm clot. With hydrochloric acid there is no immedi-
ate flocculation, but fine, white particles have begun to form within
5 minutes, and they fill the space occupied by the fluid within an
hour; the next day they are coherent and the tube can be held
horizontally without loss of water.

When formaldehyde, osmium tetroxide, potassium dichromate,
or acetic acid is added to the albumin sol, no coagulum is formed.
(Formaldehyde solution usually contains methanol, and very
sparse, fine particles are then formed in the fluid.)

It must not be thought that non-coagulant fixatives are neces-
sarily without effect on albumin, simply because they produce no

REACTIONS OF FIXATIVES WITH PROTEINS. I 33

visible result in this experiment. It can easily be shown, for in-
stance, that both formaldehvde and osmium tetroxide can render
albumin non-coagulable by ethanol ^^ (see Appendix, p. 320). It
has been known for many years that osmium tetroxide can set
undiluted egg-white into a gel, without coagulation. ^^ The ability
of osmium tetroxide to make protein sols into gels naturally
depends on the concentration of the protein. Thus 12% serum
albumin is gelled but 8% is not.^^^ Different proteins also differ
considerably, serum globulin being more easily gelled than
albumin, and fibrinogen than globulin. ^"^^ Such gels are generally
opaque. They have a tendency to liquefy eventually. '*^^' ^^

Although potassium dichromate does not coagulate albumin
sols, it does gradually render undiluted egg-white more viscous
and eventually transforms it into a weak, semi-transparent gel.
(See Seki.*^^ Potassium dichromate was used in the form of
Miiller's fluid, ^^^' ^^^ that is, with the addition of sodium sulphate.)
Acetic acid has no such effect.

There is unfortunately no substance that will represent the
fibrous proteins of the tissues so conveniently in test-tube experi-
ments as albumin will represent the globular ones. Gelatine gel
(25% w/W) can be used. It is convenient to cast the material in
15- or 30-grain pessary moulds. IVIost fixatives will not stabilize
these gels in such a way that they retain their form when put in
warm water. Gels that have been left for 24 hours in a solution of
acetic acid or potassium dichromate dissolve in water at 37° C in
a quarter of an hour or a little more. After fixation in methanol,
ethanol, picric acid, mercuric chloride, or chromium trioxide, the
gels resist complete solution for an hour or more but eventually
dissolve. Formaldehyde and osmium tetroxide contrast strongly
with these fixatives, for the gel does not dissolve, even if kept in
water at 37" C for days. The gel fixed by osmium tetroxide is
black and swells slightly in the warm water; that fixed by formalde-
hyde retains its original appearance, and one would not guess that
it had been fixed.

Interesting information can be obtained from experiments with
gelatine gels containing an admixture of albumin. Instructions for
preparing gelatine/albumin gel are given in the Appendix (p. 314).
The gel is cast in pessary moulds. It will serve for certain purposes
as a crude model of protoplasm. The refractive index is about
1*365,^^^ which is within the range shown by the protoplasm
of ordinary cells. Thus the proteins are at about the same
c

34 FIXATION

concentration as in the living cell, and both globular and fibrous
ones are present.

Gelatine/albumin gels are stabilized against warmth by fixatives
that will not stabilize gelatine alone. If gels that have stood in
fixatives for i8 hours be rinsed and transferred to a large volume
of water at 37° C, two hours later they will present the appearances
recorded in table i.

TABLE I

The appearances shozvn by gelatine j albumin gels soaked in various fixatives
for 18 hours and then left for 2 hours in zvater at 37° C^^

Form of gel

Coagulant fixatives

methanol ....

Original form roughly maintained

ethanol

,, very roughly maintained

acetone

> > > > >> > >

nitric acid

Flattened at bottom of vessel

hydrochloric acid

Shapeless mass at bottom of vessel

picric acid .

Original form roughly maintained

mercuric chloride

> > ) > ) 5 >>

chromium trioxide

> > >> ) > » »

Non-coagulant fixatives

formaldehyde

Original form exactly maintained

osmium tetroxide .

> > > >_ ) » > >

potassium dichromate .

Completely dissolved

acetic acid ....

) >

The appearances will be the same after four days. (The gel fixed
with osmium tetroxide was not observed beyond one day.)

Certain facts stand out from the experiments with gelatine and
gelatine/albumin gels. The most obvious relate to the non-coagu-
lant fixatives. It is clear that two of these (acetic acid and potas-
sium dichromate) do not fix these two proteins, in the circum-
stances of the experiment; the other two (formaldehyde and
osmium tetroxide) give much the best fixation of all the substances
tried. The strong mineral acids, though coagulant fixatives, give
very feeble stabilization of form. The other coagulant fixatives
roughly stabilize the form of the gel.

The reactions of most fixatives with nucleoproteins are very
different from those with albumin. For the pioneer work on this
subject, see Berg.^^' ^^ Convenient experiments of the same nature
are described in the Appendix (p. 317). The results of such experi-
ments are recorded in table 2.

REACTIONS OF FIXATIVES WITH PROTEINS. I

35

The table shows that ahhough picric acid, mercuric chloride,
and chromium trioxide are coagulants of both albumin and nucleo-
protein, yet these two proteins are not always coagulated by the

TABLE 2

The reactions of fixatives ivith nucleoprotein solution.^^

(See Appendix, p. 317.)

Result

hmnediate

Next day

Coagulajit of fiucleo-
protein

acetic acid

Thick coagulum

Precipitate has fallen and
occupies bottom one-
third of tube

nitric acid
picric acid

mercuric chloride

chromium trioxide .

Coarse coagulum

Fine flocculi, gradually

thickening
Fine flocculi

Coagulum

> > >> >>
>> > > >>

Fine flocculi remain sus-
pended

Precipitate has fallen and
occupies bottom half of
tube

Interynediate

hydrochloric acid

formaldehyde .
methanol

Fluid whitens (becomes
slightly opaque) ; no
visible coagulum

No coagulum

> >

A small precipitate (only
a few mm deep) has
fallen to bottom of tube

Extremely minute floc-
culi have formed

No coagulum, but the
fluid has become much
more viscous

Non-coagulmit of nucleo-
protein

ethanol .

osmium tetroxide
potassium dichromate

No coagulum

>>
>>

No coagulum (except thin

skin on surface)
No coagulum
>>

same substances. Thus acetic acid, a non-coagulant of albumin, is
a precipitant of nucleoproteins, a fact that must be correlated with
its almost invariable inclusion in fixatives for chromosomes.
Conversely, ethanol coagulates albumin but not nucleoprotein.
Potassium dichromate coagulates neither.

When gelatine/albumin gels are acted upon by fixatives, they
generally either shrink or swell. The results of a series of

36 FIXATION

experiments carried out with gels cast in pessary moulds are
shown in fig. i. The fixatives acted for 18 hours. Those fixatives
that are not aqueous solutions are seen to shrink the gels, while
the acids, especially acetic acid, swell them strongly; the two
saturated aqueous solutions (picric acid and mercuric chloride)

FIG. I. Graphical representation of the changes in

volume undergone by gelatine/albumin gels during 18

hours in various fixatives used at the concentrations

shown on p. 24.^*

change the volume least. The swelling eff'ect of acids is progressive.
A simple aqueous gelatine gel (15% w/W) shows the effect well. A
gel cast in a pessary mould swelled in acetic acid solution to about
13 times its original volume in a week.

Gelatine/albumin gels placed in distilled w^ater increase in
volume by about 40% in 18 hours. If sodium chloride is added to

REACTIONS OF FIXATIVES WITH PROTEINS. I 37

the water at |, i, 2, or 4%, the swelUng is almost exactly the same
as in distilled water.

Different fixatives penetrate into protein gels at different rates.
Our understanding of the subject dates from Medawar's investiga-
tion ^^^ of the rate of their penetration into coagulated blood
plasma. In his experiments the fresh blood of cocks was cleared of
corpuscles by centrifuging and poured into tubes closed at one
end; the plasma in the tubes was then coagulated by the addition
of a trace of tissue-extract. The tube was placed in a large volume
of fixative. The rate of penetration of coagulant fixatives was easily
noted, since the plasma became opaque on coagulation. A sharp
line divided uncoagulated from coagulated plasma. To test non-
coagulant fixatives, Medawar dissolved indicators in the plasma;
these showed the progress of the fixative into the plasma by change
of colour.

The experiments showed that in penetrating into a protein
gel, fixatives obey the laws of diffusion. If ^is the distance pene-
trated, t is time, and K a constant depending on the fixative used,
then

d = K./t.

It is convenient to measure d in millimetres and t in hours. It
follows that K represents the distance in mm penetrated in one
hour.

When indicators are used to measure the penetration of fixatives,
what is being measured is the progress of the fixative at the con-
centration necessary to give a visible colour-reaction with the
particular indicator used. There is no reason to suppose that this
will be the same as the concentration necessary to fix the protein.
The following experiments were designed to overcome this
difficulty. ^^ For full practical details, see Appendix (p. 318).

Gelatine/albumin was chosen as the protein gel into which the
fixatives were to penetrate. The gelatine gave the necessary
solidity, while the albumin marked the progress of coagulant
fixatives by becoming opaque. Small tubes were filled with the gel.
A rubber pipette-bulb closed one end, while the other was left
open (fig. 2). The tube was placed in a large volume of fixative, in
which it floated vertically. The penetration of coagulant fixatives
could easily be measured from time to time. A diamond-mark
near the top of the tube served as reference-point. The distance

38 FIXATION

penetrated, d, is a-b in fig. 2. It is to be noted that the distance
measured is not necessarily the same as the depth of the fixed
protein: that would only be so if the process of fixation caused no
swelling or shrinkage.

A

B

DIAMOND
MARK

FIXED GEL

UNFIXED GEL

FIG. 2. Pipettes used in the measurement of the

rate of penetration of fixatives into gelatine/

albumin gel.^^

A, the pipette filled with gel, before being placed in the
fixative. B, the pipette after immersion for a period in a
fixative. If the fixative is a protein-coagulant, the line of
demarcation between unfixed and fixed gel can be seen,
c, the glass tube has been turned upside down and the
unfixed gel has run out.

The distance the fixative has penetrated is a-b.

The measurement of the rate of penetration of noncoagulant
fixatives is more complicated. When the fixative has acted for a
particular time {i), the tube is lifted out; the pipette-bulb is
removed and placed on the other end of the tube. The tube is then
floated in water maintained at 37° C. The unfixed gel soon melts
and runs out; the fixed part remains in the tube (fig. 2, c).

Acetic acid fixes neither gelatine nor albumin, and its rate of
penetration can therefore not be measured with gelatine/albumin

REACTIONS OF FIXATIVES WITH PROTEINS.

39

gel. If nucleoprotein be substituted for albumin, however, the
limit of fixation is clearly seen. For the method of preparing
gelatine/nucleoprotein gel, see Appendix, p. 315.

Since potassium dichromate is not a fixative of gelatine, albumin,
or nucleoprotein, this method cannot be used to measure its rate
of penetration.

It is convenient to make observations at 2-, 3^, 4^, and 5^ hours,
and so on, if desired, up to 12- hours. If the distance penetrated is

HOURS

FIG. 3. Graph showing the rate of penetration of fixatives into gelatine/

albumin gel (acetic acid into gelatine/nucleoprotein gel). The fixatives

were used at the concentrations shown on p. 24.^^

plotted as ordinate against an abscissa divided into equal parts
representing o to i, i to 2^, 2^ to 3^, 3^ to 4^, and 4^ to 5^ hours,
etc., the results can be shown as lines that would be straight if the
equation d = K^/t were exactly obeyed. It will be seen from
fig. 3 that the lines are nearly straight. It is probable that they
would have been even more nearly so if the temperature of the
room had remained constant. The only substance that does not
show general obedience to the equation is osmium tetroxide. The
fixed gel in this case appears to offer some resistance to penetration,
for the curve falls with the passage of time. Up to 4^ hours, the
^- value is fairly constant at i-o, but from 4^ to 12^ hours only
0-31.

The values of K are shown below. The figures are based on

40

FIXATION

observations at 5^ hours. Apart from those for osmium tetroxide,
they would not have differed much if periods other than 5^ hours
had been chosen instead.

hydrochloric acid
nitric acid
formaldehyde .
acetic acid

K

4"65

4-3

3-6

2-75

mercuric chloride
methanol

chromium trioxide
osmium tetroxide

2-2

1-45
I-o

0-85

picric acid

0-8

It is to be remembered that acetic acid penetrated into a some-
what different gel from the others.

It follows from the equation that a fixative with a K value of
I-o (chromium trioxide) will penetrate 20/x (the diameter of a large
cell) in I -44 sec. ; that is to say, it penetrates that distance at the
rate of 50 mm per hour, but the rate falls off so rapidly that in fact
it only penetrates i mm in an hour, and it takes 100 hours to pene-
trate I cm. As we shall see (p. 68), penetration into tissues is
slower than into gelatine/albumin gels, and it is obvious that the
internal parts of pieces of tissue several cm thick cannot be
effectively fixed even by the most rapidly penetrating fixatives.

The results with gelatine/albumin gel agree in general with
Medawar's, so far as coagulant fixatives are concerned, but fixa-
tives penetrate more quickly into coagulated blood-plasma than
into the gel used in the experiments described here.

We turn now from naked-eye observations to microscopical
study.

The minute structure of protein coagula was first investigated
by the German botanist, Berthold,^^ in 1886. He believed that the
protoplasmic network seen in preparations of plant cells was a
coagulation artifact. He put a drop of egg-white on a slide and
noticed the formation of a microscopical network in it on the
addition of water (presumably by the coagulation of globulin and
ovo-mucoid). He also noticed the formation of separate granules on
the addition of an aqueous solution of iodine.

Another German botanist, Schwarz,^^^ who was aware of
Berthold's findings, published the results of a much fuller investi-
gation in the following year. He added absolute ethanol, picric

REACTIONS OF FIXATIVES WITH PROTEINS.

41

acid solution, tannic acid solution, and Flemming's fluid to
aqueous solutions of dried egg-white at various concentrations,
and studied the resulting material under the microscope. He
noticed that if the solution of egg-white was dilute, fine granules
were seen, while with higher concentrations these joined together
to form a network of fibres. He made similar observations with
peptone and soluble gelatine (probably metagelatine). His figures
of the networks produced in soluble gelatine by the action of
tannic acid are reproduced here in fig. 4, A, b. Schwarz considered

B

s*.^

X

[

V,

>^v ••»..

- 1 J' V --^ .

.^ - -.-\ J ^ \}.-\ . \

\ is, ■ \.^ .t

V'w .\-.

FIG. 4. Protein coagula seen under the microscope.
Each scale represents lo/n.

A, dilute solution of soluble gelatine, fixed by 0-5 % tannic acid.
B, ditto, fixed by 6'^o tannic acid. C, egg-white fixed by mercuric
chloride (saturated solution in o-6°o sodium chloride); paraffin
section, i/x or less. D, ditto, fixed by potassium thiocyanate. (a
and B from Schwarz; ^'"^ C and D from Hardy. ^")

that the granules or fibres lay in a continuous Grundsubstanz .
They were more colorable by picric acid than was this interstitial
material.

Schwarz's important work has been very much overshadowed
by Hardy's,^^^ and indeed the Cambridge physiologist carried the
investigation a good deal further. He worked chiefly with egg-
white, but also with gelatine. He caught up a drop of protein solu-
tion in a loop of silk thread and placed it in an aqueous solution of
mercuric chloride or some other fixative ; he then usuallv embedded
it in paraffin and cut thin sections (down to o-6/x). He also used
frozen sections and minute, teased fragments of unembedded

42 FIXATION

material. As fixatives he also used the vapour of osmium tetroxide
and the heat of steam.

Tvi^o of Hardy's figures are shown in fig. 4, c, d. He found that
weak protein solutions generally showed separate microscopical
particles after fixatives had acted, while stronger ones resulted in
the appearance of a sponge or net. As the figures show, the net was
thickened at the nodal points. The size of the meshes varied with
the fixative used. The vapour of osmium tetroxide (a non-
coagulant fixative, as we have seen) was alone in not producing a
microscopically visible structure.

Hardy's main contribution was his discovery that there is no
Grundsiihstanz. The whole of the coagulum consists of granules or
spongework, with water in between. This was evident when suffici-
ently thin sections were cut, for there was nothing in the meshes of
the net that could be demonstrated even by saturated solutions of
dyes. In thicker sections the out-of-focus appearance of other
parts of the net gave the misleading appearance of an interstitial
substance, but Hardy realized that in finished microscopical
preparations the meshes were occupied only by Canada balsam or
other mounting medium. Thus, as he said, the essence of fixation
by coagulant fixatives is the separation of fluid from solid, of
water from protein ; and the solids, if sufficiently abundant, hang
together to form a network (or in some cases a honeycomb-like
structure). As a result, water can often be squeezed by hand from
a fixed protein, though a pressure of 400 lb to the square inch will
not separate it from an unfixed protein gel. When water has been
separated from protein, it can be replaced by other fluids. While
the water is still present, there is usually little change of volume ;
but when the replacement occurs, there is usually shrinkage.
Similarly a silicic acid gel shrinks w^hen its water is replaced by
another fluid.

It has already been remarked (p. 22) that the network pro-
duced by the action of coagulant fixatives has the same refractive
index as dry protein, and that is why mounting media having about
the same r.i. as dry protein give such glassy transparency to
microscopical preparations of the tissues of organisms.

Hardy considered that osmium tetroxide in aqueous solution
produced a network in egg-white, but the effect was more prob-
ably due to the water of the solution acting in the absence of salt
on globulin.

It might be thought that the structure produced in protein sols

REACTIONS OF FIXATIVES WITH PROTEINS. I 43

and gels by coagulant fixatives would have very deleterious effects
in microtechnique. So long, however, as the network is a very fine
one, the effect is not wholly damaging, for the spaces produced by
coagulation give access to embedding media, especially paraffin,
that would otherwise be unable to enter. It is a striking fact that
the great majority of the familiar fixative mixtures contain a
coagulant (see p. 148).

CHAPTER 3

The Reactions of Fixatives with
Proteins 2. The Chemical Changes

The most familiar fixative substances will be considered one by
one, from all points of view connected with their use as fixatives,
in chapters 5 and 6 (pp. 89 and iii). Their chemical composition
and the ions they form when dissolved in water wdll be mentioned
there. To prevent unnecessary repetition, this information will
not be given in the present chapter.

It is important to explain certain terms and conventions that
will be used in this chapter and throughout the rest of the
book when reference is made to the chemical structure of pro-
teins.

A protein has two constituent elements, which will be called the

NH
HCH glycine

NH

I
HC.CH.,SH cvsteine

r°

NH

HC(CH2)4NH2 lysine

c=o

A small part of a protein chain. The atoms of the
backbone are shown in bold letters.

backbone and side-groups. The backbone is made up of those parts
of the amino-acids that are the same in all. These are repeated

44

REACTIONS OF FIXATIVES WITH PROTEINS. 2 45

over and over again, like vertebrae; they articulate through the

O

II
peptide link, — C — NH — . The backbone is folded in various

ways; this folding will not be represented in the structural
formulae used. The side-groups are those parts of the amino-acids
that distinguish one from another. These project laterally from the
backbone, like ribs. They alternate in direction, but in this book
they will all be represented as projecting to the right. This con-
vention will be adopted partly for economy of space, partly because
the different side-groups are more easily recognized if always
written in the same sequence (just as words are more easily read
if the sequence of the letters is not reversed).

A sequence of linked amino-acids (backbone and side-groups)
will be called a chain. The word molecule will not be used loosely.
It seems inapplicable when protein chains are linked together by
chemical bonding through their side-groups to form net-like struc-
tures of indefinite size, perhaps extending from one end of a cell
to the other.

A substance cannot act as a fixative if it attacks the peptide link.
Far from being proteolytic, fixatives tend to link protein chains
together and thus give mechanical stability.

An important feature of the structure of proteins is that a
particular side-group may react chemically without necessarily
changing the nature of the chain as a whole. Suppose, for instance,
that a particular protein containing very few cysteine side-groups
is exposed to a substance that reacts with nothing in the chain
except -SH groups. Obviously the chain as a whole will be scarcely
affected, and no fundamental change in the nature of the protein
will have occurred. If a particular side-group is capable of reacting
with a particular dye, it will retain this property when a fixative
has only blocked some other side-groups.

Despite these facts, the appearance and 'nature' of a protein,
especially its solubility in water, are often markedly changed by
fixation, and the substance is often said to be 'denatured'. Un-
fortunately this word has been used vaguely, and with different
meanings by different authors. So long as it referred to a loss of
solubility, one knew what it meant ; but loss of solubility is accom-
panied by increase of reactivity, and this fact has altered the mean-
ing entirely: for if the increase in reactivity occurs, the process is
often called denaturation, even though solubility is actually

46 FIXATION

increased. It is obvious that fixation cannot involve increase in
solubility, and there are therefore many 'denaturing agents', such
as urea, that could not possibly be fixatives. It is easier to study
denaturation, however, if the product be soluble; and for this
reason much of our knowledge of the process, admirably sum-
marized in several reviews, ^^' ^^' ^^^' *^^ is not directly applicable
to the problems of microtechnique. For similar reasons most of the
study of denaturation has been devoted to proteins in the form of
sols, yet gels are more interesting to the biologist, because proto-
plasm is essentially a soft gel; and gelled proteins can also be
denatured.

We may broadly distinguish additive from non-additive fixation.
In the former, the fixative molecule or a considerable part of it
adds itself to the protein; in the latter it does not. The word 'de-
naturation' was formerly held to imply that the change involved
was non-additive, but this usage is not quite general today. It has
even been said that any chemical change in a protein, not involving
disintegration into amino-acids, is denaturation.^^^ In this book,
however, the word will be used to mean a non-additive change in
a protein, causing it to become less capable of remaining in
intimate relation with water as a sol or gel, and more reactive. The
resulting loss of solubility ordinarily manifests itself in coagulation,
if the protein be a sol ; a gel is rendered harder and opaque.

Polypeptides, in the sense of short chains of amino-acids, cannot
be denatured: the process occurs with the very long chains of
amino-acids that constitute proteins.

The chief non-additive or denaturing fixatives are these:
methanol, ethanol, acetone, nitric acid, hydrochloric acid.

If a protein sol be mixed with a denaturing fixative, the reaction
is usually so quick that coagulation appears to be instantaneous.
Careful experiment has shown, however, that there are in fact
three stages. First, reactivity is increased; then flocculation
follows, but the flocculus is soluble in weak acids or alkalis;
finally the flocculus hardens into a coagulum, only soluble by
proteolysis. Some so-called denaturing agents, such as urea, only
cause the first change; and it is for that reason that they are not
usable as fixatives.

Denaturation may be brought about in many different w^ays; for
instance, by subjection to very high pressure, extension in ex-
tremely thin films, or exposure to ultrasonic waves or ultra-violet

REACTIONS OF FIXATIVES WITH PROTEINS. 2 47

light. Freezing and thawing can cause denaturation, especially of
lipoproteins, though freezing- drying does not: indeed, freezing-
drying should be used instead of fixation if undenatured protein is
needed. The only means of denaturing that are used in routine
microtechnique are heat and certain chemical agents; the former
is seldom used except for the fixation of blood-films.

We cannot establish for each protein a particular temperature of
denaturation. Most proteins are denatured if held in water at
60° C for a long time, though ribonuclease is extremely resistant.
If a soluble protein be dried, heated to 100° C, and cooled, it
retains its solubility. Thus heat alone does not suffice to denature:
it is hot water that causes the reaction.

Methanol, ethanol, and acetone are the chief organic substances
used in microtechnique as denaturing fixatives. They ally their
eff"ects to that of heat; or, to put it in other words, the reaction has a
high thermal coefficient. A very low concentration of ethanol
suffices to denature proteins if the temperature is above 60° C;
at —8° C a concentrated solution produces a flocculus, but this is
soluble in water at room temperature; ^^^ there is no measurable
denaturation of any sort below — 15° C. (It must be mentioned
that alcohol actually favours the solution of a few proteins, such as
zein and gliadin.) The alcohols and acetone dehydrate strongly,
but it is clear that more than this is involved in denaturation, since
mere drying is ineffective.

Hydrochloric and nitric acids are denaturing fixatives, but they
are not very commonly used. Denaturation by heat or other means
occurs rather readily at the iso-electric point of each protein; at a
slightly more or less acid pH than this the tendency to denature is
less; from about pH 2-5 downwards and again above pH 10,
denaturation occurs at room-temperature. The strong mineral
acids at about o-^N are quite useful coagulant fixatives. In con-
centrated solution they disintegrate proteins into amino-acids.
Acetic acid does not ionize freely enough to produce a pH suffici-
ently low to coagulate proteins; in addition, the acetate ion inter-
feres with denaturation (p. 64).

We must picture the proteins in the tissues of the living organism
as maintaining a special relation with w^ater through their various
water-soluble groups. This is so whether the proteins be globular
or fibrous, and if the latter, whether bound into gels or not. Since
the protein molecule is very long, the amino and carboxyl groups
of the terminal amino-acids are not of much significance in this

48 FIXATION

respect, and indeed if the polypeptide chains are bound together
into a gel, there need not be any terminal amino-acids. The chief
hydrophil constituents are the carboxyl, hydroxyl, and amino-
groups of the side-chains, and the carbonyl of the backbone; the
imino-groups of several amino-acids, the amido-group of aspara-
gine, and the sulphydryl of cysteine can also associate with water.

NH

HC(CH,)2C(

\OH

c—

NH

glutamic acid

HC.CHo— / \0H

tyrosine

c—

NH

HC(CH2)4 NH2

c=o

lysine

Part of a protein chain. The hydrophil groups are shozvn in bold letters.

The most striking effect of a coagulant fixative on a protein sol
or gel is an alteration in the effects of these hydrophil groups,
which formerly held water so firmly that it could not be separated
by powerful mechanical force, but now can be squeezed out by
hand (p. 42). The effect is irreversible, for the protein loses its
power to imbibe water and resume its former properties.

To the chemist the most significant result of denaturation is the
increase in reactivity. Certain reactive constituents of the protein
were previously present, wholly or partly, in latent form: now they
exhibit themselves and respond freely to tests for their presence.
An increased digestibility by enzymes results, and indeed accounts
for the fact that we cook our protein food. Two views have been
held about the nature of the latency of the reactive groups before
denaturation. It has been suggested that they do not exist in the
natural protein, but originate during the process of denaturation
(and also in proteolysis). Most students of the subject, however,
consider that the reactive groups are present in the original pro-
tein, but something hinders the access of test-reagents to them.
They may be involved in linkages between protein chains or

REACTIONS OF FIXATIVES WITH PROTEINS. 2 49

between different parts of the same chain, or simply inaccessible
because the chain is so tightly folded.

The reactive groups liberated by denaturating agents are all
ionizing side-chains of amino-acids. Chemists have given most of
their attention to those reactive groups that are particularly easily
shown by simple tests. It is probably for this reason that they have
concentrated so much on the sulphydr}d (-SH) of cysteine, the
disulphide (-S-S-) of cystine, the phenyl of tyrosine, and the
indolyl of tryptophane. It seems that the sulphydryl group, which
has been particularly carefully studied in relation to denaturation,
does not arise in this process by the splitting of disulphide bonds.
Its emergence from latency gives reductive properties to the protein.

The groups that become reactive on denaturing are of great
importance to the histochemist, but in general microtechnique we
are especially concerned with the amino and carboxyl groups, for
these provide the main points of attachment for acid and basic
dyes respectively (see p. 167); and since we usually colour cyto-
plasm and nuclear sap with acid dyes, we are above all interested
in the reactivity of the amino-groups of lysine and arginine.
Unfortunately, not very much attention has been paid to the re-
lease of these particular groups from their latent condition in the
natural protein. This is partly due to the fact that their appearance
is not so dramatic, since some of them are readily accessible in the
protein before denaturation.

If there were a special freeing of basic groups from a latent
condition, the protein would become more reactive with acids and
acid dyes; and conversely. Such changes would produce a shift
in the iso-electric point of the protein. Denaturation by alcohols
produces less change in the iso-electric point than fixation in
other ways, and that is why these substances are chosen as fixa-
tives when we want to study the basicity or acidity of proteins by
the simultaneous use of acidic and basic dye-ions (p. 262). In
general, there is a small shift (roughly pH 0-5) of the iso-electric
point in the less acid direction on denaturation.

When a conjugated protein (lipoprotein or nucleoprotein) is
denatured, the protein constituent commonly separates from the
substance with which it was combined, and the latter then reveals
its presence more readily.

The specificity of proteins, especially any immunological
property, is generally irreversibly lost by denaturation. Trypsin,
however, can be reversibly denatured: it loses and regains its

D

50 FIXATION

power to digest. As a rule enzymatic properties are destroyed, and
special fixatives must be chosen if they are to be displayed in
microscopical preparations. If the right denaturing agent is chosen,
it will coagulate the proteins in general but leave certain enzymes
more or less intact. Sections may then be cut and placed in a solu-
tion of a suitable substrate. The latter must be carefully chosen,
for it must leave microscopically visible evidence of the places in
the tissue in which it was attacked by the enzyme : the product of
the reaction must be immobile and either visible or capable of
being made visible. In this roundabout w^ay the original site of
the enzyme can be determined. It is chiefly for work of this sort
that acetone deserves to be listed as a fixative. Certain enzymes,
such as alkaline phosphatase, are resistant to its action. Acetone
distorts tissues seriously and would never be chosen for purely
morphological studies. It has, however, certain uses as a fixative
in other branches of histochemistry beside enzymology.

An important eflfect of denaturation on globular proteins is
their elongation into fibres. This change can best be witnessed
when loss of solubility has not yet occurred. The extension of the
protein molecules renders the sol more viscous, and since they
tend to arrange themselves parallel with one another in a moving
fluid, the formerly isotropic sol now exhibits the birefringence of
flow. The protein never extends fully into a straight chain on
denaturation, but always remains to some extent folded. Remark-
ably enough, the fibrous proteins are aflFected in the opposite way:
the chain shortens somewhat by folding. There is thus an approxi-
mation of both kinds of proteins towards a similar structure, but
this is fibrous, not globular. A coagulum could not be formed if
the product of denaturation had not been fibrous. The firmness of
the coagulum must depend on the nature of the bonds that tie
the fibres together.

A microscopical preparation of denatured protein generally
shows a network that appears to consist of interlacing fibres, fused
where they touch; there may or may not be swellings at these
points. The appearances are similar to those shown in fig. 4 (p.
41). The difference in scale between these microscopically visible
fibres on one hand and the polypeptide chains of the proteins on
the other is enormous; still, the latter underlie and make possible
the former. The visible network is an artifact, but it reminds us of
an important truth about the submicroscopical structure of pro-
teins. In protein gels we must imagine the polypeptide chains

REACTIONS OF FIXATIVES WITH PROTEINS. 2 51

keeping a small and fairly regular distance from one another, with
water every\vhere intervening between one and the next except
where they actually adhere. When denaturation has taken place,
the water is no longer firmly bound, and the polypeptide chains
can come close up against one another to form microscopically
visible strands, while microscopically visible spaces are formed
between one such strand and another. A network of strands is thus
formed. If globular proteins are present, these will extend on
denaturation and participate in the formation of the strands.

It may be said in brief summary that fixation by denaturing
renders globular proteins fibrous; irreversibly alters the relations
of all proteins with water, so that this is no longer firmly held; and
greatly increases the reactivity of certain side-groups of the
constituent amino-acids, so that they respond much more readily
to tests for their presence and attach themselves freely to dyes ; but
the specificity of many proteins is lost. The chemical denaturing
agents are non-additive: they achieve their effects while remaining
essentially detached.

Many fixatives are additive to proteins. The difference between
additive and non-additive fixation is not quite so great as might
be supposed. Most substances undergo a profound change if an
atom be added to them, but proteins are necessarily different in
this respect. A fixative might be capable of adding itself to the
side-group of a particular amino-acid that was very scantily
represented in a particular protein; the fixative might nevertheless
denature the protein as a whole. For this reason one must not
draw too sharp a distinction between denaturation by non-
additive fixatives and the changes brought about by additive ones.
The distinction is partly a matter of the arbitrary use of words.
Many of the changes described in the preceding part of this chap-
ter occur also when certain additive fixatives react with proteins.

Nevertheless, a valid distinction does exist in most cases.
Further, certain additive fixatives, unlike any non-additive ones,
are non-coagulant of albumin. The familiar additive fixatives may
be grouped thus: —

coagulant non-coagulant

mercuric chloride formaldehyde

chromium trioxide osmium tetroxide

picric acid

52 FIXATION

These substances will now be considered in turn.

The reactions of mercuric chloride and other mercuric
salts with proteins have been carefully studied by several
authors. ^^^' ^*^' ^*^' ^^^ The metal becomes attached to protein in
several different ways at the same time, unless special precautions
are taken to isolate the reactions.

Mercuric salts react with the sulphydryl (-SH) group of the
cysteine component of proteins. If the amount of mercury be
restricted to what will combine with the sulphur present in the
protein, no other reaction will occur; this may therefore be called
the primary reaction. It is possible to isolate a fraction of serum
albumin that contains only one sulphydryl group in each mole-
cule. ^^^ This may be crystallized in the form of a compound with
mercury, which contains one atom of mercury to two molecules
of the albumin fraction. The evidence suggests strongly that
mercury forms a link between cysteine residues. Each of the latter
is potentially mercurium captans, a mercury-catcher, ready to
form a compound (mercaptide) with the metal.

"?

NH NH

I
CH2.S-Hg-S.H2C.CH

I

Q=o c=o

. . ^^ .

Mercury forming a link between cysteine side-groups in tzvo protein chaitn

Many such links could be formed between protein chains in
which cysteine occurred repeatedly, and many chains could be
bound together into a single, polymeric whole. This would tend
towards coagulation.

It is unlikely that mercaptide-formation is the main cause of
coagulation. When an excess of mercuric salt is present, as in
ordinary fixation, much more of the mercury is taken up in other
ways, to which we must now turn our attention.

The uptake of mercury by proteins is profoundly affected by
acidity and alkalinity. The main features of the process are set out
schematically in table 3. A scale of pH is not provided, because
each protein would require a different one. The lines dividing the
degrees of alkalinity and acidity must not be regarded as separating
sharply the several reactions, for in fact these overlap, so that more
than one reaction may occur at any particular pH. The table calls
attention to the reactions that are dominant in certain broad regions
of alkalinity and acidity.

REACTIONS OF FIXATIVES WITH PROTEINS. 2

53

TABLE 3

Diagrammatic representation of the chief reactions of mercuric chloride
with proteins, in the presence of excess of mercuric chloride

Mercury is
taken up as

Nature of bond

Strength
of bond

Coagulation

STRONG
ALKALINITY

Hg -

To amino-groups
by secondary
valencies ; to car-
boxyl groups by
main valencies

Firm

Little tendency
to coagulation

WEAK
ALKALINITY

neutrality —

WEAK
ACIDITY

iso-electric"|
point of >
protein J

HgCla

To amino-groups
by secondar\-
valencies

Ven,^
loose

Opposed by
sodium chlor-
ide

HgCl4]" 1 To amino-groups
by main valen-
cies

Very
loose

Promoted by
sodium chlor-
ide

STRONG (No reaction except with sulphur
ACIDITY of cysteine)

Little tendency
to coagulation

The reactions of mercuric chloride with protein are affected by
the abihty of mercury to form bonds through subsidiary valencies.
For instance, mercuric chloride can in certain circumstances react
with ammonia to form a diammine.*"^ This results from the fact
that mercury can accept extra electrons from donor atoms.

HsN^ /CI

HaN-^ ^Cl
Mercuric chloride combined with ammonia

In Strongly alkaline conditions, the mercuric ion is taken up by
the amino-groups of proteins (on the side-chains of lysine and
arginine), by bonds of this sort.^^^ The mercury then reacts as

"?
1

NH

(CH2)4NH.,

;=o

Lysine forming part of a protein chain

though present as mercuric hydroxide. The mercuric ion may
perhaps combine in a similar way with the -NH.CO- groups of
the protein backbone. Beyond this, the mercuric ions form

54 FIXATION

salt-linkages with the carboxyl side-groups of certain amino-
acids.

These reactions appear not to have a strongly coagulative effect,
but the mercury is firmly held. This can easily be shown by test-
ing with mercuric potassium iodide, Hgl2.2KI, a useful reagent
for distinguishing between mercuric ions or loosely held mercury
on one hand, and the firmly held metal on the other. The reagent is
yellow, but gives the red colour of mercuric iodide in the presence
of the ions or loosely held metal.

A different kind of combination is characteristic of weakly
alkaline, neutral, or weakly acid conditions down to the iso-
electric point of the protein. The mercuric chloride is taken up as
a whole molecule through subsidiary valencies, mainly by the
amino side-groups of lysine and arginine, and is now held very
loosely, so that a red reaction is given with mercuric potassium
iodide. The formation of this loose compound is associated with
coagulation. Coagulation is opposed by sodium chloride. This is
because the chloride ions have a stronger affinity for mercuric
chloride than the protein has. The anion [HgCl4]" is formed. ^^^ If
albumin is coagulated by mercuric chloride and washed, the clot
is reaily dissolved by a saturated solution of sodium chloride (or
potassium iodide).

On the acid side of the iso-electric point of the protein, but still
within the range of weak acidity, the ion [HgClJ" combines
through main valencies with the amino-groups of the protein,
which are ionized to some extent on the acid side of the iso-
electric point. The compound formed is again very loose, but
coagulation is now promoted by the presence of sodium chloride,
which increases the amount of the reactive mercuric ion.

In conditions of strong acidity mercuric chloride does not react
with proteins, except to form mercaptide ; there is little tendency to
coagulation.

In practical microtechnique, acids and sodium chloride are
often added to solutions of mercuric chloride, apparently without
much consideration of the complex consequences. Tissues that
have been fixed with mercuric chloride are sometimes placed in
Lugol's solution. This is likely not only to decompose the mer-
captide with oxidation of the former sulphydryl groups to di-
sulphide,^^^ but also to dissolve the coagulum.

If mercuric chloride were to block all -NHg groups by combining
with them, but left the -COOH groups untouched, the protein

REACTIONS OF FIXATIVES WITH PROTEINS. 2 55

would become very acidic; conversely, if it were to block all
-COOH groups, the protein would become very basic. These
changes would control the reactions with dyes, for the coloured
ions of the latter associate with the acidic and basic groups of
proteins (see p. 192). It is rather a strange fact that most authors
who have considered mercuric chloride in this connexion have
taken it for granted that this salt simply blocks -COOH groups
and thus makes the protein more basic (that is, more attractive to
'acid' dyes (p. 167)).^^^' *^^' ^64 it is stated, however, by the Ameri-
can histochemist Gomori ^^^ that mercuric chloride reduces the
attraction of proteins for acid dyes, by blocking -NH2 groups.
As we have seen, the reaction of this fixative with proteins is in
fact very complicated.

Mercuric chloride is pre-eminent among fixatives for leaving
tissues in a condition conducive to brilliant dyeing. It is relevant to
consider here the characters of a fixative that will achieve this end.
It must not fix proteins in such a tight gel that dyes cannot pene-
trate, but must somehow make them porous. It must knock DNA
off from combination with protein and precipitate it in a form in
which it will associate readily with basic dyes. It must leave the
protoplasmic proteins in a state in which they will accept acid
dyes, so that these parts may be given a colour that will contrast
with that given to the DNA. It is evident that mercuric chloride
achieves these ends, in the circumstances of ordinary fixation;
but we await a full explanation of its superiority over other fixatives
in these respects.

Most fixatives leave proteins in a digestible state; denaturation,
as we have seen (p. 48), promotes digestibility. In experiments
of this sort the excess of fixative must be very carefully washed
out: for instance, by washing in running water for 20 days.
When this has been done, proteins fixed by additive fixatives
generally remain digestible. It has been shown, however,
that mercuric chloride slows down slightly the rate of digestion
of the proteins of blood plasma by pepsin and trypsin.^^® This
may be due to a change in the protein, but it is also possible
that some of the bound mercury transfers itself to the enzyme
and fixes it.

Picric acid is an alkaloidal reagent: that is to say, it precipitates
alkaloidal salts from solution in water. This is a usual property of
large, complex anions. Free amino-acids combine with the anion

56 FIXATION

to form picrates ; each amino-acid gives a crystal of characteristic
form. For a plate showing the crystals of eleven picrates of amino-
acids, see Schmidt. ^*^ Picric acid resembles other alkaloidal
reagents in coagulating soluble proteins, but the chemistry of
the process has not been worked out. The obvious point of
attack would be the amino side-groups of lysine and arginine,
but if all these were blocked, the protein would lose most of
its affinity for acid dyes. In fact, however, picric acid gives
egg-white a strong affinity for acid dyes, but scarcely any for
basic ones.*^^ It seems probable that acid dyes are able to re-
place picric acid at its points of association with amino-groups.
It is to be recollected that picric acid is not only a fixative but
also a dye (p. 185), and that dyes can replace one another in
this way. The reduction in affinity for basic dyes has not been
explained. Picric acid also forms additive compounds with
phenols, and combination with the side-group of tyrosine is not
excluded.

Chloroplatinic acid is another alkaloidal reagent. The incorrect
name of platinum chloride disguises the fact that the metal forms
part of a complex anion, [PtClg]^.

The mode of action of chromium trioxide on proteins in the
process of fixation is not well understood. We have a considerable
amount of knowledge about the reactions at high temperatures,
because they have been studied by the textile chemists. ^^^' ^^^' ^^^
These reactions must be briefly mentioned here, though their
relevance is doubtful.

Compounds of chromium are used in the pre-treatment of wool
before the application of dyes. The chemistry of the process of
'mordanting' will be dealt with in chapter 11. In industry, sodium
dichromate is commonly used for the purpose, often with the
addition of acid. When a dichromate is dissolved in water, the
anions produced are essentially the same as those produced by
chromium trioxide (see pp. 105 and 126). The chromium is anionic,
and it makes little or no difference whether chromium trioxide or
acidified dichromate is used. Industrial mordanting is carried out
at boiling point, and the fibre is often treated afterwards with a
reducing agent.

Anionic chromium appears to associate itself chiefly with those
side-groups of the wool proteins that contain -NHg and -OH,
and with the -CO.NH- group of the protein backbone. Accord-
ing to the textile authorities, the metal is taken up partly as

REACTIONS OF FIXATIVES WITH PROTEINS. 2 57

anions, but some of it also transforms itself into a non-ionic,
sexi-covalent form, some of the six links being with the groups just
mentioned. ^^^

The industrial process results in the firm binding of chromium
to protein. When wool is simply steeped in the mordant at room-
temperature, however, this is not so.^^^ The fibre takes up the
mordant and becomes yellow, but the metal is loosely held and
can be washed out by a buffer at pH 8.

In microtechnical fixation chromium trioxide is used at room-
temperature, yet the metal is firmly bound and cannot be removed
even by prolonged washing. ^^^ Solutions of chromium trioxide are
strongly acid (p. 105), and simple denaturation by acidity might be
thought to be partly responsible for the results; but proteins are
much more violently coagulated than by hydrochloric or nitric
acid, and are so altered that they cannot be digested by pepsin or
trypsin. ^^^ Less is known about the chemical changes underlying
the action of this fixative on proteins, in the circumstances of the
fixation of tissues in microtechnique, than about the changes
underlying the action of any other common coagulant fixative.
If the reaction were largely with the amino-groups of the proteins,
as is supposed to be the case in the mordanting of textile fibres,
these groups would presumably be blocked and no longer available
to ordinary acid dyes; yet this fixative, perhaps above all others, is
favourable to the action of such dyes (pp. 109 and 204). As an
oxidizing agent, it is likely to react with the -SH of the cysteine
side-group ; also with the phenyl of tyrosine, the indolyl of trypto-
phane, and the iminazol of histidine. It has recently been shown
that prolonged fixation in solutions of chromium trioxide does in
fact interfere with histochemical tests for the three last-named
amino-acids.^^-^

A considerable amount of misunderstanding has arisen from
the fact that chromium trioxide is used in the tanning of leather. It
has been supposed that the process throws light on fixation by
chromium trioxide in microtechnique. Unfortunately this is not
so, for anionic chromium plays no part in tanning. The chromium
trioxide is reduced to a basic salt, and it is cationic chromium that
reacts with the proteins of skin to make leather. ^^^ The reaction of
chromic sulphate with proteins has also been investigated chemi-
cally,^^ but this again is irrelevant to our subject, for the same
reason. Cationic chromium is seldom used deliberately as a fixa-
tive in microtechnique, but it may be formed by the reaction of

58 FIXATION

chromium trioxide with the tissues, and may then perhaps itself
react with the tissues.

We turn now to the non-coagulant additive fixatives, formalde-
hyde and osmium tetroxide. These are of particular importance,
for the very fact that they are non- coagulant makes them unlikely
to distort tissues and cells seriously. It must nevertheless be kept
in mind that subsequent treatment, especially embedding in
paraffin, often results in gross distortion.

Formaldehyde can be caused to form compounds with various
amino-acids,^"^ but most of these reactions appear to be irrelevant
to microtechnique. The compound with tyrosine, for instance, is
only formed by heating for several hours in acid solution. *^^

The reactions of formaldehyde with polypeptides, each con-
sisting of chains of only one particular amino-acid, are extremely

NH NH NH

HCH HC(CH2)2C\ HC(CH2)2C(

I I ^OH I \NH2

Glycine Glutamic acid Glutamine

The amifio-acids of poly glycine, polyglutamic acid, and polyglutamine

instructive. Experiment shows how much formaldehyde such
polypeptides will remove from solution. ^"^ Polyglycine binds very
little formaldehyde; so does polyglutamic acid. Polyglutamine, on
the contrary, binds more formaldehyde than any other macro-
molecule, so far as is known.

Silk-fibroin consists mainly of alanine and tyrosine. Like poly-

NH
HC.CH3 alanine

:—o

NH

HC.CHaC^ ^ OH tyrosine

f

=0

?

Part of a molecule of silk-fibroin

glycine and polyglutamic acid, it binds very little formaldehyde:
less than one-twentieth as much as polyglutamine.

REACTIONS OF FIXATIVES WITH PROTEINS. 2 59

These facts suggest strongly that formaldehyde reacts with the
-NH2 groups of proteins. Lysine is largely involved. ^'^^' 177,203

NH NH NH

I I I!

HC(CH2)4NH2 HC(CH2)3NH.CNH2

r

Lysine (left) and arginine {right), as components of proteins

The side-group of arginine also reacts, but only above pH 8,
a degree of alkalinity unusual in microtechnical fixation. This
reaction is not fixative, for it does not stabilize the protein; indeed,
it increases susceptibility to swelling by acids (p. 64) and
shrinkage by high temperature. ^^^

When proteins are deaminized by the substitution of -OH
groups for -NHg, their capacity to bind formaldehyde is much
reduced. The work on this subject has been done largely with
collagen and casein, because the toughening of these substances
by formaldehyde is important in industry. The physical changes
associated with tanning by formaldehyde do not occur after
deamination.

Formaldehyde could react with lysine by simple addition, with
formation of the side-group -(CH2)4NH.CH20H, or alternatively
the reaction could be a condensation to form -(CH2)4N=CH2 and
water. The former reaction would be a special case of the general
equation RH + CH20=RCH20H. The -OH in the additive com-
pound is reactive, and a methylene bridge, R — CH2 — R-"^, is thus
easily formed. Formaldehyde is commonly thought of as a reducer,
but it is to be noted that in this particular reaction it acts on the
contrary as an oxidizer. This was pointed out long ago by Kings-
bury,^^^ who did so much to help to place fixation on a scientific
basis.

A methylene bridge between lysine side-groups on two previ-
ously separate protein chains at once suggests itself; but the
stoichiometric relation between lysine and formaldehyde is nearer
I : I than 2:1, and it is therefore probable that the linkage is
between the lysine of one protein chain and a different group con-
taining nitrogen in another protein. This could easily be glutamine.
A link between lysine and the nitrogen of the peptide (-NH.CO-)
link of the main protein chain is also possible. It will be remem-
bered, however, that the peptide link of polypeptides is scarcely

6o FIXATION

reactive, for polyglycine and poly glutamic acid bind very little
formaldehyde. Nylon also binds very little formaldehyde, ^^^
though the -NH.CO- group occurs repeatedly in the mole-
cule.

' \C(CH2)2C]

f

HC(CH2)4N— CHa— N^

I H H C=0

?

A methylene bridge (bold type) linking tzvo protein chains through lysine (left)

and ghitamine (right)

Bridges of these kinds would tend towards gel-formation in
proteins, or to the strengthening of pre-existent gels; coagulation
does not result. In general, cross-linkages of any kind between
protein chains result in brittleness, loss of elasticity, reduction in
the ability to bind water, and, in the case of soluble proteins,
lessened solubility."^^

Zein is interesting in this connexion. The mechanical strength
of fibres and films made of this protein is considerably increased
by the action of formaldehyde, yet it contains no lysine (and very
little arginine). It must be presumed that links are formed between
the nitrogen of an amide (-CO.NHg) group in one protein chain
with a similar group in another, or possibly with the nitrogen of a
peptide link.^'^

The reaction between proteins and formaldehyde is remarkably
slow and contrasts strongly in this respect with the quick ionic
reactions of some fixatives. In experiments with soluble proteins
and polypeptides, carried out at 70° C, only one-half of the total
amount of formaldehyde that eventually combined was taken up
in 8 hours ; after the lapse of 24 hours one-tenth of the amount still
remained unbound. ^^* In practice, fixation by formaldehyde is
presumably nearly always incomplete, except when tissues are
not only fixed but stored in a solution of formaldehyde. That is
why tissues remain capable of taking up a limited amount of acid
dyes.

The eff'ect of pH on the reaction between proteins and formalde-
hyde is complex. The reaction wdth amide groups is said to be
promoted by acidity, ^^^ but that with simple amino-groups is
antagonized ; the total effect is a slowing down, and the reaction is

REACTIONS OF FIXATIVES WITH PROTEINS. 2 6l

very slow below pH 3.^^^ The greatest binding of formaldehyde
occurs at slight alkalinity (pH 7-5 to 8).^^^

The importance of formaldehyde as a tanning agent for pro-
teins in industry has ensured a careful study of its mode of action.
The other important additive, non-coagulant fixative has no
industrial applications of this kind, since it is far too expensive for
practical use. This is unfortunate, for osmium tetroxide is one of
the most valuable fixatives in microtechnique. In electron-
microscopy it is pre-eminent on account of the faithfulness of its
fixation and the ability of chemically bound osmium atoms to
scatter electrons and thus make an image possible. It should be
noticed that osmium tetroxide could not fulfil the latter function
if it were a non-additive fixative. A mere deposition of unbound
osmium would be useless, and indeed as misleading as it often is in
light-microscopy.

That osmium reacts with unsaturated lipids (olein and oleic acid)
was known long ago to Altmann,^ but our understanding of its re-
actions with other tissue-constituents has grown slowly and is still
very imperfect. In this chapter we are concerned only with those
reactions that throw light on the way in which it fixes proteins.

The first relevant discovery was made in 1920 by Dutch chem-
ists,^^ who showed that osmium tetroxide could be used to convert
cyclohexene to cyclohexane-diol. Two points must be noticed

Ho Ho

Hof >,H H/ ^H.OH

H,l I'H Hoi Jh.OH

H2 H2

Cyclohexene Cyclohexane-diol

here: the reagent acts at the two ends of a double bond, and the
product of reaction is a compound with two adjacent hydroxyl
groups (a diol).

Reactions of this sort, whether caused by oxides of manganese

.H

\ \ /

CH 0<x C— 0\

II + ^0s02= I ;os02

CH O^ C— Q/

H

The reaction of osmium tetroxide at the ends of a double bond

or of osmium, may proceed in two stages. First, the double bond
disappears and an additive compound is formed; then a second

62 FIXATION

reaction may occur, with production of a diol and osmic acid. (It
may be remarked in passing that the expression osmic acid is here
used correctly. See p. 119.)

\ /" \ /"

C— 0\ COH

I /OsOa + 2H2O - I + H2OSO4

/C^O-^ COH

H / ^H

Diol Osmic acid

That osmium tetroxide could react in this way with many ring-
compounds was shown by the German organic chemist, Criegee,
in 1936.^^^ One such substance is indene, which is related to indole,

NH

HCCH

2

H V ^ H

Ittdole Tryptophane as part

of a protein chain

a constituent of tryptophane. It follows from work on related
substances ^'^ that the point of attack of osmium tetroxide would
be the two ends of the double bond shown here in the five-
membered pyrrol ring of indole and tryptophane.

Osmium tetroxide is capable, in certain circumstances, of
joining molecules together. This was shown by Criegee and his
associates. ^^^' ^^^ They found that osmium tetroxide acted most
readily in this way when it had first combined with pyridine, but
there are reasons for supposing that other organic substances
might take the place of pyridine. It is clear that osmium can form a
link between two ring-compounds by joining with both of them

^C^O\ II /O-^C
I )Os( I

H H

Osmium acting as a bridge between tzvo ring-compounds. (The latter are shown

only in part.)

in the way previously explained. It is tempting to suppose that
the formation of gels from soluble proteins (or of firm gels from
weak ones) may result from the ability of osmium to form bridges
of this kind between protein chains. It is remarkable that such gels

REACTIONS OF FIXATIVES WITH PROTEINS. 2 63

are often temporary (see p. 33). This may be connected with the
fact that osmium tends to free itself from combination with ring-
substances, while transforming these into diols.

Many amino-acids react with osmium tetroxide; this can be
seen by the development of a dark colloidal reaction product, or
sometimes a dark precipitate. It is doubtful, however, whether all
amino-acids that react when free necessarily do so when they are
constituents of a protein. Thus, the basic amino-acids, lysine and
arginine, are reactive when present in proteins ; but the protamines,
which contain a high proportion of basic amino-acids, scarcely
react with osmium tetroxide at room-temperature.^^ Tryptophane,
histidine, and cysteine react especially strongly when free, forming
dark precipitates.^^ Histidine, like tryptophane, possesses a double
bond between carbon atoms; the nature of the reaction with
cysteine has not been worked out. Among the various proteins, the
most reactive are those that contain a high proportion of these
three amino-acids.^^ The higher the amount of tryptophane in a
soluble protein, the lower the concentration at which that protein
can be gelled by osmium tetroxide. *^^

The anomalous non-coagulant fixatives, potassium dichromate
and acetic acid, remain to be discussed. They are anomalous be-
cause they appear not to be fixatives of ordinary proteins.

At the end of the last century Fischer ^^^ showed that potassium
dichromate did not coagulate albumin or globulin. We saw in
chapter 2 that it does not render gelatine gels insoluble in warm
water nor coagulate nucleoprotein. All this is radically altered if
the solution be acidified: the reactions are then the same as though
we had used chromium trioxide (see p. 106). We are concerned
here with unacidified dichromate. The reaction with protein is
slow at room-temperature. The unacidified salt is sometimes used
in preparing wool for subsequent dyeing, but the process is carried
out at a high temperature and the chemistry of it is unlikely to
throw much light on fixation. In ordinary microtechnical use un-
acidified potassium dichromate seems to do little to proteins be-
yond making them somewhat more basic: that is to say, more
capable of being coloured by acid dyes.^^^ There is no good evi-
dence that it has a definitely fixative effect on simple proteins,
and nucleoproteins it positively dissolves. It is, however, one of
the few fixatives that make certain lipids capable of resisting
solution during embedding in paraffin. This is almost certainly the

64 FIXATION

cause underlying the continued use of potassium dichromate in
microtechnique (see p. 128).

Acetic acid behaves quite differently. The ionization constant
being low (i-8 X io~^), the hydronium ion is not present in suffi-
cient abundance to cause denaturation, but is able to exert its
swelling effect. Indeed, this effect will be more readily produced if
proteins are not coagulated by denaturation. Collagen fibres are
swollen most by acids at pH 2-5,^^* and this is near the pH of 5%
acetic acid (see p. 134). Acids will tend to break the salt-links
between the chains; that is, the links binding together an amino-
group in one with a carboxyl group in another. The breakage will
expose hydrophil groups of atoms, and it is the entry of water
attracted by these groups that is the cause of swelling.^**

Weak organic acids swell wool much more than mineral acids do
at the same pH. This is thought to be due to the action of the
undissociated part of the acid, which tends to attach itself to
amide groups through hydrogen bonds. The amide groups on

r°

HC^R

I
NH • • 0=(

=?

HCR NH

C=0 HCR

Parts of tico protein chains held together by a hydrogen botid (...) between
two amide groups. The latter are indicated by bold lettering.

neighbouring protein chains are supposed to associate with one
another through hydrogen bonds, and the breakage of these by un-
dissociated acid would permit swelling. The fact that acetic
acid has much more capacity to swell proteins than mineral acids
have is well shown in fig. i (p. 36).

In addition to the hydronium ion and undissociated acid, there
is the acetate ion to consider. This has a separate effect in fixation,
which has been carefully studied by the American botanist
Zirkle.^^^ He found that all acetates, including hydrogen acetate
(acetic acid), tend to act similarly at the same pH (except that the
salts of heavy metals show the effects of the cation rather than that
of the anion). The acetate ion does not fix ordinary proteins;
indeed, it opposes the action of denaturing agents. *^^ It can react,
however, with nucleoproteins. Whether it does so or not depends
on whether the pH is on the less or the more acid side of pH 4-4

REACTIONS OF FIXATIVES WITH PROTEINS. 2 65

(or thereabouts). On the less acid side (for example, the unacidified
sodium or ammonium salts), the acetate ion is not fixative, but
macerates badly; on the more acid side there is coagulation of
nucleoprotein, or separation of DXA from combination with
protein with immediate precipitation.

No attempt will be made to give a general account of the fixation
of lipids and carbohydrates, because these substances are not
stabilized by most fixatives. The reactions of particular fixatives
with them will be mentioned in chapters 5 and 6.

^

^ . >r

'>-'^'rv,

^'^i<Ariy I 3U,

CHAPTER 4

The Reactions of Fixatives with
Tissues and Cells: Methods of

Research

Flemming ^^^ claimed that osmium tetroxide produced a net-
work in the Zellsaft of Spirogyra. In making this statement in 1882,
he appears to have been the first person to mention the production
of a microscopical network in the tissues of an organism through
the action of a fixative. This was a strange eflPect to attribute to
osmium tetroxide, the very last fixative one would choose to pro-
duce a network. Hardy,-^^ however, also described fine-meshed
nets in paraffin sections of tissues fixed with solutions of osmium
tetroxide; the vapour left the protoplasm homogeneous. As a
general rule the non-coagulant fixatives leave tissues more or less
transparent, apart from any extrinsic artifact they may themselves
deposit. This is because they do not produce a microscopic net-
work. Subsequent dehydration or other treatment may cause
opacity.

The coagulant fixatives usually make tissues opaque, and white
or yellowish: that is to say, the fixed material reflects light but
does not readily transmit it (unless special steps are taken to cause
transparency). The origin of the new surfaces responsible for this
optical change in the tissues was first disclosed by Berthold,^^
who in 1886 described the coagulation of the protoplasm of plant
cells by alcohol and other reagents. He realized clearly that the
coagulation of protoplasm was essentially the same as that of egg-
white. Similar observations on plant cells were made by
Schwarz,*^^ who supposed in error that coagulation was the separa-
tion of the firmer from the more fluid parts of the proteins of the
protoplasm. It was Hardy ^^^ who showed that the net was the
whole of the coagulated protoplasm, the meshes being filled with
nothing but the mounting medium in the finished preparation.

66

I cm

MERCURIC
CHLORIDE

PICRIC
ACID

CHROMIUM
TRIOXIDE

\:2^'.

..5 *

v

i ^

FIG. 5.

A, lobes of the li\er of the rabbit left for 24 hours in fixatives (at the concentrations
shown on p. 24) and then cut across.^ '^ B-E, photomicrographs illustrating Young's experi-
ments on the addition of indifferent salts to fixatives. The cells are neurones in the stellate
ganglion of Sepia officinalis. B, fixed in 4",, formaldehyde dissolved in distilled water; c,
in the same dissolved in sea-water. D, fixed in Champy's fluid made up w'ith distilled
water; E, in the same made up with sea-water.

(b-e are reproduced by kind permission of Prof. J. Z. Young and of the Editors and
Publishers of Nature.)

REACTIONS OF FIXATIVES WITH TISSUES AND CELLS 67

Hardy worked with the intestinal epithehum of the woodlouse
Oniscus (fig. 6) and with various cells of the frog and mammals.
'On the whole', he remarked, 'living cell-substance does react to
fixatives just as does solid or
fluid soluble colloid.' By solid
or fluid colloid he meant pro-
tein gels or sols.

Since tissues become opaque
when invaded by coagulant
fixatives, it is possible to ob-
tain information about their
rate of penetration. If mam-
malian liver or some other
rather homogeneous organ be
chosen, the line of demarca-
tion between fixed and unfixed
tissue is often extraordinarily
clear and regular (fig. 5, a),
though the fixative runs more
quickly along the loose tissue
accompanying large blood-
vessels than through compact
masses of cells. It makes little
difference whether excised
blocks of liver-tissue be used,
or whole lobes; the latter,
however, give the clearest
results. It is evident that the connective tissue sheath of the organ
forms a negligible barrier.

The earlier work on this subject ^^^' ^^^' ^^"^ was done before
Medawar ^^"^ had disclosed the principles governing the diffusion of
fixatives into protein gels (p. 37). Experiments on the penetration
of coagulant fixatives into whole liver-lobes of the rabbit prove
that the same principles govern penetration into tissues. ^^ It is
only necessary to suspend liver-lobes by threads in a large volume
of the fixative under test, and to remove these at measured inter-
vals and cut them across. The thickness of the coagulated tissue
may be measured rather accurately. The figure obtained is not
necessarily the same as the distance penetrated into the fresh
tissue, because the fixative may change the volume of what it

FIG. 6. A cell from the intestine of Onis-
cus (woodlouse), fixed by mercuric
chloride (saturated solution in o-6°o
sodium chloride); I'4jli paraffin section,
dyed with iron haematein: to show the
coagulation of protoplasm.
(From Hardy.213)

i

68

FIXATION

coagulates. Mercuric chloride is convenient in this respect, for it
does not greatly alter the volume of mammalian liver (see p. 76),
The results with this fixative are shown in fig. 7. The approxima-
tion of the graph to a straight line is remarkably close. It is clear
that the rate of penetration falls oiT with time exactly as it does
when fixatives enter clotted blood-serum or gelatine/albumin gel.
The rate of entry is, however, considerably slower. Thus mercuric

FIG. 7. Graph showing the thickness of rabbit-liver fixed by
a saturated aqueous solution of mercuric chloride in various

times. ^^

chloride penetrates 4-2 mm into liver in 25 hours (K = 0-84), but
1 1 mm into gelatine/albumin gel (K = 2-2). A survey of the work
done in this field shows that the rate of penetration of coagulant
fixatives into gelatine/albumin gels is in general agreement with
their rate of penetration into tissues, except that the latter is much
slower. Picric acid, however, goes more slowly than chromium
trioxide into the gel, but more quickly than the trioxide into liver
(fig. 5, a). (The reader will remember that in all such experiments,
the concentrations of the fixatives were those listed on p. 24; see
p. 32.) It is not obvious why fixatives should go so much more
slowly into tissues than into the gel, for the latter contains about
the same concentration of protein as ordinary protoplasm (p. 33).

REACTIONS OF FIXATIVES WITH TISSUES AND CELLS 69

It seems probable that the hpids of the cell-membranes act as
barriers.

Fixatives penetrate more quickly into small than into large
pieces of tissue. This is of practical importance. It might be
thought a matter of indifference whether a large or a small piece
were cut out of an organism for fixation: attention could be con-
centrated on the edge of the large piece, which would be as well
fixed as the smaller. This is not so, for the following reason.

Imagine two blocks of tissue, with opposite sides parallel. One of
them, block A, is exposed to the fixative on one side only (say the
lower side): the other block, B, is exposed on the two opposite
sides.

Consider first the passage of the fixative into A. It may be re-
garded thus. The fixative instantly reaches the upper side at
infinitesimal concentration ; later at progressively higher concentra-
tions. For a considerable time the concentration at the upper side
will be below that at which protoplasm is fixed (that is, either
coagulated or changed into a stable gel). Meanwhile the substance
will be gradually penetrating at its fixative strength, according to
its K-value, and the line separating the fixed from the unfixed
tissue will be advancing at continually diminishing rate towards
the upper side, in accordance with the equation given on p. 37.

In block B the course of events will be different. On each side
the substance would tend to penetrate at its fixative concentration
at the same rate as it did on one side of block A, but from the first
moment onwards there will be an infinitesimal contribution from
the opposite side, and this will continually increase. The concen-
tration at any given place at any given time will therefore be higher
than it would have been if only one side had been exposed to the
fixative. The thinner the piece, the more evident this effect will be.
It follows that a fixative will penetrate more quickly into a small
than into a large piece of tissue, and small pieces should therefore
be used unless there is some particular reason for not doing so.

It is instructive to watch cells being fixed, and to record the
effects not only on the ground cytoplasm, but on every constituent
part. It was probably the transparency of living cells that held back
this kind of investigation in the early days of microtechnique. As
one optical method after another has been invented to overcome
this difficulty, so enthusiasts have hastened to apply the new
microscopes to problems of fixation. Not everyone who has

yo FIXATION

engaged in this work has provided himself with the pre-requisite
knowledge of the subject of fixation. It has seemed sufficient to
put living cells under one of the kinds of microscopes that evade
the difficulty of transparency, and simply watch what happens
when the fixative is added. It is not always realized that this is a
test of preservation, not of fixation. It has already been remarked
(p. 28) that the classical work of this kind was done by Strange-
ways and Canti,*^^ by dark-ground microscopy.

The most convenient cells are those that will flatten themselves
against a coverslip and adhere firmly to it. The amoeboid meso-
dermal cells of various kinds that move outwards from cultured
tissue-fragments fulfil these requirements well.*^^' ^*' ^^*' ^^^ Their
thinness makes them readily observable by dark-ground or phase-
contrast microscopy, and their adherence makes it easy to replace
the culture-fluid by a fixative. The replacement can be achieved
by quite simple apparatus,^^^ but a special perfusion-chamber has
been designed ■^*- that w^ould lend itself well to this kind of work.

The extreme thinness of cultured amoeboid cells, when
stretched over the surface of a coverslip, presents disadvantages.
Such cells are far from being representative of cells in general, and
their firm attachment and unusual shape prevent them from
shrinking, or at any rate from showing clearly that they shrink
(apart from the retraction of pseudopodia). Loose cells have their
merits, despite the technical difficulties they present. Among those
that have been used are salivary corpuscles, ^^^ polymorphs, *^^
cultured neurones,-"*^ and amphibian spermatogonia.^^*

The results obtained by research of this kind will be incor-
porated briefly in chapters 5 and 6. By the common consent of all
engaged in it, osmium tetroxide gives the most faithful preserva-
tion of the living condition of the cell (fig. 8, A, b). Chromium
trioxide may be cited as an example of a fixative that alters the
structure considerably (fig. 8, c, d). The pseudopodia are blunted;
cytoplasm and nuclear sap coarsely coagulated; lipid droplets
fused (probably by the stresses of coagulation); mitochondria
destroyed. In dividing cells, however, the chromosomes are
rendered clearer than they were in life.

One would wish to obtain quantitative information about the
chemical changes wrought by fixatives on tissues and cells. A
piece of tissue may be analysed chemically, as a whole, before and
after fixation,^^^ or the fixative solution may be analysed for dis-
solved tissue-constituents after fixation, *^^ but the substances

\v>.i

I ■ i

\

w •* /

D

FIG. 8. The effect of fixatives on cultured cells from the chorioid or
sclerotic coats of the eye of the chick embryo.

A, living cell. B, the same cell in 2"o osmium tetroxide solution, c, living cell.
D, the same cell in 0-5 '^o chromium trioxide solution. Dark-ground microscopy.
Drawings by Dr H. Fell.

(From Strangeways & Canti,*" by kind permission of Dr H. Fell and the
Company of Biologists, Ltd.)

i

REACTIONS OF FIXATIVES WITH TISSUES AND CELLS 71

revealed in these ways may be hard to trace to their sources. An
estimate of the amount of nucleic acids lost by cells on fixation
can be obtained by measurement of the optical density of parts of
the cell before and after fixation. ^*^ This method depends on the
opacity of nucleic acids to ultra-violet light of wave-length
265 m[i. A photomicrograph may be taken with this light before
and after fixation, and the density of the images compared.
Allowance must be made for change in volume of the part
measured. Results with cultured fibroblasts from the chick embryo
suggest that 4% neutral formaldehyde may reduce the amount of
nucleic acids (DNA -j- RNA) in the nucleus by between 10% and
35%. It would be interesting to have comparable figures for all
primary (unmixed) fixatives.

In order to investigate fixative as opposed to preservative action,
it is necessary to examine cells that have been through the com-
plete routine of microtechnique, and to compare these with living
cells. Buchsbaum ^* in Chicago has worked in this way with cul-
tured amphibian macrophages. He studied them in life by phase-
contrast microscopy, and then fixed, washed, dyed, and mounted
the same individual cells, making photographic records at every
stage. The results showed that the changes caused by fixation
were more fundamental, in this particular case, than any that re-
sulted from subsequent treatment; but embedding was omitted,
and this is often a destructive process.

All the studies of this kind so far mentioned are open to one
major objection. The cells were separate from one another, and the
fixative came directly up against them at its full concentration.
In ordinary microscopical preparations the superficial cells are
often damaged while those lying below the surface are well fixed
(p. 28). Unless it so happens that information is particularly
required about separate or superficial cells, the test should be
designed in such a w^ay that its result does not depend on the
response of such cells, and it should involve the whole of the after-
treatment that is to be used in practice. Since dyeing does not
ordinarily introduce serious distortions, any dye or dyes that will
reveal the structure of the object clearly may be used; but for a
full test of any fixative it would be necessary to try all the usual
embedding and mounting media.

Ideally each primary (unmixed) fixative should be tried with
each embedding and mounting medium, and each fixing/embed-
ding/mounting method should be assessed from the point of view

72 FIXATION

of general micro-anatomy and histology, and also separately for
its results with each cellular constituent (cell outline, ground
cytoplasm, mitochondria, lipid globules, nuclear membrane,
nuclear sap, nucleoli, chromosomes, etc.).

A comprehensive study of this kind has never been made, but
a certain amount of work has been done. More than half a century
ago Tellyesniczky 497-499 jj-^ Budapest studied the spermatogonia
and spermatocytes of the salamander in paraffin sections of testes
that had been fixed in various ways. Unfortunately he deliberately
omitted to make careful comparisons with the living cell. 'So far as
possible', he wrote, 'we avoid the question of Lebenstretie.^ ^^"^
The Austrian cytologist Pischinger *^^ studied the effects of various
fixatives on the nuclei of mammalian liver, with subsequent em-
bedding in paraffin. His results, though interesting, w^ere marred
by his belief in the homogeneity of living nuclei.

For any test of this sort, it is of great importance to choose a
suitable test-object. There are some organs that are reasonably
well fixed even by indifferent fixatives. The intestine of the newt is
an example. Others are susceptible to distortion in various degrees.
One needs an organ that is difficult to fix well, so that the defects
of fixatives may expose themselves clearly. It seems likely that
protoplasm is easily fixed when it contains a high proportion of
protein, and conversely; but this can only be proved when the
interference microscope has given more information about the
protein-content of cells. The distinguished cytologist, Belar,^^
particularly recommended the testes of grasshoppers as test-
objects for fixatives. The former are only available during a limited
season, but crickets {Acheta domesticiis) are convenient laboratory
animals, ^^^ and their testes are ripe at all times of year. (See

P- 329-)^

As Belar remarked, the testis of the laboratory mouse is ein sehr

heikles Object for tests of fixatives. The kidney-cortex of the same
animal is equally sensitive. A test of fixatives has recently been de-
vised ^^ in which these two organs are the objects of study. The
test involves the use of only a single embedding medium and a
single mountant, and the results are judged only from the point of
view of the histologist. The test is made as thorough as these
limitations permit. It could easily be made wider in scope.

The testis is cut into four parts and the kidney cortex also into
small pieces. After appropriate washing the fixed tissues are
passed through graded ethanols and toluene into paraffin wax.

REACTIONS OF FIXATIVES WITH TISSUES AND CELLS 73

This method of embedding was chosen partly because it is so
much used, partly because it has a strong tendency to distort and
thus provides a stringent test. Sections are cut at 8/x and dyed v^ith
Hansen's haematein (so-called Trioxyhdmatein).^^^ When a piece
of tissue has been dehydrated, passed through toluene or other
antemedium, and embedded in paraffin, it has probably undergone
all the distortion that the processes of microtechnique can w^reak
upon it, and the choice of mounting medium is therefore of little
significance for the purpose of the test. Canada balsam was chosen,
mainly because it seems to remain the most popular mountant.

It is to be wished that we had some means of estimating the
quality of fixation objectively. The degree of shrinkage or swelling
can be measured (p. 75), but we have no other numerical data
and for the present it is necessary to rely on subjective impressions.
Anyone who proposes to judge fixatives should equip himself
for the work by prolonged experience in the study of living
cells.

People are often prejudiced in their beliefs about the value of
different fixatives. To prevent this from influencing the results of
tests, it is essential that the judge should never know what fixative
he has been judging until after he has given his opinion. He
should examine many preparations fixed in different ways and
report fully on each before being told which is which. Preparations
fixed in fluids that contain osmium tetroxide are usually darkened,
and they should therefore be bleached before dyeing, so as not to
be recognizable.

Written records should be made under a standardized set of
headings before a definite opinion of the value of a fixative is
formed. The following will serve as examples of headings in tests
carried out with the testis of the mouse: —

outlines of seminiferous tubules (whether smooth as in life, or
wrinkled by shrinkage) ;

spaces betw^een tubules (whether artificially enlarged or dis-
torted) ;

cohesion of spermatogenetic cells (whether maintained or lost) ;

cytoplasm of spermatogenetic cells (whether homogeneously
fixed, coarsely coagulated, or disintegrated) ;

chromosomes (whether fixed in life-like form in the meiotic
phases) ;

dyeing (whether intense or weak, differential or diffuse).

74 FIXATION

The report on the kidney-cortex of the mouse may be made
under these headings: —

spaces between convoluted tubules (as above, under semini-
ferous tubules) ;

cytoplasm of convoluted tubules (as above, under spermato-
genetic cells) ;

free border of epithelium of first convoluted tubules (whether
smooth or ragged);

nuclei (whether smoothly rounded or distorted) ;

red blood corpuscles (whether they retain their natural shape or
are swollen into spheres or otherwise distorted) ;

dyeing (as above).

The most delicate parts of the two organs are the cytoplasm of
the primary spermatocytes and the free border of the first con-
voluted tubules. Really good fixation of these is rarely seen in
paraffin sections.

It is convenient to compare fixatives by assigning them to differ-
ent grades. It is an arbitrary matter to decide the number of such
grades, but everyone who undertakes work of this kind will agree
that two are too few (because more accurate distinctions can be
made with confidence) and ten too many (because, if the assessment
were repeated with the same slides, it would often happen that the
same preparation was not assigned to the same grade). It is perhaps
reasonable to make five (grade I the best, grade V the worst). To
prevent waste of time from prolonged indecision, one may some-
times record the result as I-II, II-III, etc. Examples of grade I
and grade V fixation are shown in fig. 9. Some of the results of this
test with simple fixatives and mixtures will be mentioned in
chapters 5, 6, and 7.

The fact that a fixative falls into a low grade in this test by no
means condemns it. Grade V fixatives are usable with many organs
that are less delicate than mammalian testis and kidney-cortex.
They may also have particular virtues of their own. Altmann gives
blocks of tissue that crack easily with paraffin embedding; there
is considerable shrinkage and distortion of the cells ; chromatin is
dissolved away. Mitochondria, however, are excellently fixed, and
Altmann remains a useful fluid despite its manifest defects. (The
cracking does not occur when Altmann tissue is embedded in
collodion.) As a general rule, nevertheless, it is obviously best to

B

y

lOfa

FIG. 9. Sections of the testis of the mouse, to show good and bad
fixation. Both are 8/x paraffin sections dyed with Hansen's

haematein.^^

A, fixed with Allen's 'B.15'.^ Grade I fixation. B, fixed with osmium tetroxide,
i^o, buffered at pH 7-4. Grade V fixation. (With methacrylate embedding, the
same fluid gives good fixation for electron microscopy.)

REACTIONS OF FIXATIVES WITH TISSUES AND CELLS 75

rely on fixatives in the higher grades, when the embedding and
mounting media to be used are the same as in the test.

It is wrong to speak of good and bad fixatives without mention-
ing the after-treatment. A fixative that stabiUzes structure against
embedding in paraffin is hkely to give good results with milder
after-treatment, but one that works well with mild after-treatment
may give poor results with paraffin. Flemming's weak fluid ^^^
seems a very poor fixative (grade IV-V) when paraffin sections are
used. It should be remembered that the great cytologist left
minute pieces of tissue or separate cells in his fluid for half an
hour and then examined them in water without any other treat-
ment whatever. There was no question of embedding, and he
admitted that his preparations lost some of their delicacy if
mounted in glycerine. In fact, he used his weak fluid rather as a
preservative than as a fixative. Ordinary formaldehyde/saline
(formaldehyde at 4% in 0-7% aqueous sodium chloride solution)
gives poor results (grade III-IV) with paraffin sections, but quite
good (grade II) with collodion. An extreme example of the same
kind is provided by Palade's buffered osmium tetroxide solution. ^^^
This fixative is one of the best available to the electron-micro-
scopist when tissues are embedded in methacrylate, but the results
with paraffin embedding are very poor (fig. 9, b).

Non-quantitative tests, including the one we have been dis-
cussing, have considerable value. Thus it is important to know
that one fixative destroys mitochondria, a second leaves them
undamaged but unfixed, and a third stabilizes them against paraffin
embedding. A multitude of similar examples could be quoted.
Still, it is to be wished that we had more knowledge that could be
expressed in numbers. Our main fund of quantitative information
concerns shrinkage and swelling.

The length and breadth of a whole organism may be measured
before and after fixation, and at various subsequent stages of
treatment. This can be useful in various ways (for instance, by
enabling us to judge the initial size and therefore the age of em-
bryos that have been fixed) ; ^^^ but it provides little knowledge
about the action of fixatives, since shrinkage or swelling may
affect mainly the cells or mainly the body-cavity and other inter-
cellular spaces, or a vertebral column may interfere with changes
in length that would have occurred in its absence. Soft organisms
with relatively small internal cavities do, how-ever, provide useful

76 FIXATION

information, for shrinkage is usually uneven and therefore pro-
duces obvious changes of form. The ctenophore Pleurobrachia is a
delicate organism, well suited to observations of this kind. If
fixed in a solution of formaldehyde in sea-water, it retains its
shape well. If it now be transferred to 10% ethanol and thence
slowly through 20%, 30%, 40%, and so on up to absolute ethanol,
the main shrinkage occurs in 70%, with obvious deformation.^^ The
lower ethanols, up to 60% or thereabouts, appear not to cause
much shrinkage of properly fixed tissues, but there is always a stage
in dehydration by ethanol, somewhere in the higher concentrations,
against which no known fixative will protect the tissues.

Accurate measurements may be made of changes in volume
undergone by whole organs, ^^^' ^^ or by large cubes cut from whole
organs. ^*^ The liver and spleen are especially suitable because they
are fairly homogeneous in structure and contain no large empty
spaces. The volume may be recorded when the organ is fresh and
again at any number of subsequent stages up to and including
infiltration with paraffin. The results of experiments of this sort
with liver are shown in table 4. It will be noticed that the change in

TABLE 4

The effect of fixation and subsequent treatment on the volume of whole

livers {? mammalian).
[Means of several observations in each case; rearranged from the data

of Berg. ^'^)

■

Mean volii

me expressed as
organ

% of fresh

after

after

after

dehydration

infiltration

fixation

with absolute

zvith melted

etha?wl

paraffin vcax

mercuric chloride, sat. aq. .

91

80

70

formaldehyde, 4°^ aq. .

99

83

68

chromium trioxide, 1% aq. .

78

68

64

ethanol, 96%

82

76

55

potassium dichromate, 3%

aq. .

100

64

49

picric acid, sat. aq.

74

64

42

volume cause by fixation itself gives little indication of the total
shrinkage that will have occurred when the organ has been in-
filtrated with paraffin wax. Potassium dichromate, for instance,
causes no change of volume but allows the tissue to be excessively

REACTIONS OF FIXATIVES WITH TISSUES AND CELLS 77

shrunk by after-treatment. Picric acid shrinks strongly and permits
much subsequent shrinkage. Mercuric chloride and formaldehyde
gave less final reduction in volume than any other primary (un-
mixed) fixative that wsls tested.

Hooks may be inserted in each end of a piece of liver or other
tissue, and a string from one of them may be attached to a lever;
this will record on a revolving drum the progressive changes in
linear dimensions that occur at every stage from fixation up to
embedding in melted paraffin. ^^^ Experiments carried out in this
way show that in routine microtechnique there are three stages at
which shrinkage chiefly occurs after fixation. There is sudden
shrinkage of mammalian organs on passing from 70% to 90%
ethanol (reminiscent of the shrinkage of Pleiirobrachia on passing
from 60% to 70%), a gradual but pronounced shrinkage in xylene,
toluene, or other antemedium, and a further prolonged shrinkage
in hot paraffin. (Liquid paraflin at room temperature also shrinks.)
These, then, are the particular stages in embedding against which
fixatives do not give protection. There are obvious advantages in
methods of embedding that evade some or all of the stages at
which shrinkage chiefly occurs. It is to be noted that absolute
ethanol does not have much effect on the volume of fixed tissues
that have already been soaked in 90%.

It is particularly important that we should know how fixation
and the subsequent processes of microtechnique affect the volumes
of individual cells. Those of regular form naturally commend
themselves for work of this kind, because they are easy to measure.
The eggs of cows,^^^ rabbits, ^^^ and echinoids ^^^ have been chosen
for this reason ; also the fully-grown primary spermatocytes of the
snail {Helix asper'sa).^'^^ The possible sources of error in this kind of
investigation have been best appreciated by Ross,^^^ who, in his
recent work at Oxford, has made every effort to avoid them and
has even taken into consideration the change of magnification of
objectives when mounting media of different refractive indices are
used. He has expressed his results in the form of medians and
shown the scatter of his observations by the use of frequency
polygons, while the others have relied on the means of relatively
few measurements.

The diameters of the cells can be measured while they are alive
in sea-water or other appropriate ffuid, and at any number of
subsequent stages in the routine processes of microtechnique.
Hertwig's ^^^ investigation was the most complete in this respect.

78 FIXATION

Paraffin embedding has always been used. It is desirable that
comparative studies should be made with other media.

Fixation does not necessarily shrink cells, and may indeed swell
them strongly, but dehydration always reduces the volume below
that of the living cell, and immersion in xylene carries the process
still further. Some typical examples are given in tables 5 and 6.

TABLE 5

The volwries of unfertilized eggs 0/ Arbacia pustulosa at various stages of
embedding in paraffin, expressed as percentages of the volumes zvhile alive
in sea-water. Each figure is calculated from the mean diameter of 10 to 15

eggs. [Data of Hertzvig.^^^)

Fixative

Volume in

fixative,
24 hrs.

ethanol,
70%

ethanol,
abs.

xylene

mercuric chloride, sat. aq.
formaldehyde, 4% in sea-water .
ethanol, abs. ....

130

105
48

64

70

50
SO

49

48

41

TABLE 6

The volumes of the pritnary spermatocytes of the snail, Helix aspersa, fixed

in various simple fixatives, embedded in paraffin, dyed, and mounted in

Canada balsam. They are expressed as percentages of the volume of the

living cell. {Rearranged from the data of Ross. ^'^^)

Formaldehyde, 4% aq. (neutralized)

• 34%

Mercuric chloride, sat. aq.

.30%

Chromium trioxide 0-75% aq.

29%

Acetic acid, 5% aq. ....

28%

Potassium dichromate, 5% aq.

23%

Picric acid, sat. aq. ....

20%

Ethanol, abs. ......

19%

Shrinkage always occurs when cells fixed in aqueous media are
transferred to 70% ethanol, and further shrinkage at each sub-
sequent stage, at an}^ rate up to xylene. We unfortunately have no
data on the change of volume of whole cells on passing from
xylene to melted paraffin.

A comparison of table 4 with tables 5 and 6 suggests that in-
dividual cells are more shrunken than whole organs by the pro-
cesses of routine microtechnique. It is doubtful whether most
histologists and cytologists realize the extreme degree of com-
pression that a cell has undergone when it is examined in an
ordinary microscopical preparation. (See fig. 10.)

REACTIONS OF FIXATIVES WITH TISSUES AND CELLS 79

The evidence suggests that nuclei generally shrink less than
whole cells. Some of Hertwig's results with nuclei are shown in
fig. II. It will be noticed that infiltration with melted paraffin
causes further shrinkage of nuclei bevond that caused bv dehvdra-
tion and soaking in xylene.

Shrinkage is harmful partly because it always involves distortion,
partly because structural detail that approaches the limit of resolu-

LIVING CELL

FORMALDEHYDE. MERCURIC CHROMIUM ACETIC POTASSIUM PICRIC ETHANOL,

4% CHLORIDE, TRIOXIDE. ACID, DICHROMATE, ACID, ABS.

SAT. 0-75% 5% 5% SAT.

FIG. 10. Outlines of the fully-grown primary spermatocyte of the
snail, Helix aspersa, to show the effect of fixation and subsequent
treatment on the size of the cell. All the cells except the one at the
top have been fixed, embedded in paraffin, sectioned, dyed, and

mounted in Canada balsam.

(Diagrammatic but accurately to scale: from the data of Ross.***)

tion of the microscope may pass beyond it. It is not surprising
that paraffin wax is so much favoured by microtechnicians, for
serial sections can be obtained very easily and their attachment
to slides is quick and simple ; but there is probably no other method
of embedding that involves so much shrinkage. Our familiar fixa-
tives may perhaps owe their survival to the fact that they give the
best (or least bad) results with paraffin embedding. (See p. 148.)
There is room for much experiment here. We need new fixa-
tives that will stabilize tissues better against existing after-treat-
ments, and new after-treatments that will cause less shrinkage, or
preferably none at all. The stearates of diethylene glycol present
considerable advantages in this respect. *^^' ^^^ The polyethylene
glycols ('carbowax') ^^^' ^^^'^1 are particularly promising em-
bedding media, because tissues can be passed directly into them
from water and shrinkage seems to be slight; but there are still

8o

FIXATION

practical difficulties in their use, especially in the flattening of
sections and their attachment to slides.

'For the good working of many reagents it is of the greatest
importance that they should be dissolved in a medium that
cannot itself produce disorganization by osmotic disturbances.

ETHANOL, ^^

lU

Zi 80 -

r3

<

LU

u

UJ

a.

If)
<

o

ALIVE

IN
RINGER

FIXATIVE
24 HR5.

ETHANOL,
ABS.

XYLENE

CANADA

BALSAM

AFTER

PARAFFIN

FIG. II. Graph showing the effect of fixation and
subsequent treatment on the volume of the nuclei
of cartilage-cells (scapula of Salamatidra tnacu-
losa). Each point represents the mean of 20
measurements.

(From the data of Hertwig.**")

Therefore, for instance, for the preservation of marine algae
alcoholic solutions and solutions in distilled water are to be
absolutely rejected. Parallel experiments with picric acid, osmic
acid, and iodine dissolved in alcohol, in distilled water, and in
sea-water show without exception that only the solutions in
sea-water give really good results, when one uses delicate
objects.'

These statements were published in 1882 by Berthold,^" who, as
we have seen (pp. 40 and 66), was one of the first to approach
the problems of fixation scientifically. We do not yet understand

REACTIONS OF FIXATIVES WITH TISSUES AND CELLS 8l

the causes underlying the resuhs he obtained. The facts may be
briefly stated thus. The best fixation is often obtained if the fixa-
tive substance is dissolved, not in distilled water, but in a solution
of an 'indifferent' or non-fixative salt, such as sodium chloride or
sulphate.

Miiller ^^^' ^^^ used sodium sulphate nearly a century ago, in a
mixture with potassium dichromate, and this salt is used in making
up Zenker ^^^ and Helly ^^* to this day, because these fixatives
were based on Miiller's. Heidenhain ^'^^ long ago dissolved
mercuric chloride in a solution of sodium chloride, and the same
salt is commonly used with formaldehyde.

The advantage of adding an indifferent salt is shown particu-
larly clearly by the cells of marine algae and marine invertebrates.
Young ^^^ investigated this matter with the neurones of the cuttle-
fish, Sepia officinalis. He cut the stellate ganglion in two and put
one half into a solution of a fixative in distilled water, and the
other into a solution in sea- water. The two halves were then em-
bedded together in paraffin and sectioned. When the fixative was
picric acid, chromium trioxide, formaldehyde, osmium tetroxide,
or potassium dichromate, the half fixed in the solution in sea-
water was greatly superior to the other (fig. 5, b-e, opposite
p. 67). It appeared that when the cells were placed in a solution of
the fixative in distilled water, they swelled, burst, and then col-
lapsed with serious shrinkage and distortion, though this sequence
of events was not actually observed; in the presence of sea- water
they more or less retained their shape. Fixation by mercuric
chloride and acetic acid was, however, scarcely affected by the
presence of the salts of sea-water.

Hertwig ^^^ made a quantitative study of the effect of sea- water
on the fixation of the eggs of sea-urchins. Some of his results are
shown in fig. 12. It will be seen that when formaldehyde was dis-
solved in distilled water, the eggs increased greatly in volume dur-
ing fixation, but they did not burst. Increase in volume was slight
when sea- water was used as solvent, and the eggs remained
smaller than the others throughout dehydration and in xylene.

The cells of fresh-water and terrestrial organisms are less
sensitive to the presence of indifferent salts than those of marine
algae and marine invertebrates, but similar effects have been
observed in them by Sjovall,^^^ Carleton,^^* and others. For many
references to the early literature of the subject, see Schaffer.*^^
When the vertebrate kidney is fixed in formaldehyde dissolved in

82

FIXATION

distilled water, the tubules appear shrunken in paraffin sections;
but when this fixative is dissolved in 0-9% sodium chloride solu-
tion, the epithelium of the tubules sometimes swells so much that
the lumen is almost obliterated in the final preparation. ^^^ Some
impression of the eflPect of indifferent salts on the fixation of

140

120

5^ 100

o

<

80

z 60

o
a.

<

OJ

_i
O

>

40

20 -

-I

ALIVE

FIXATIVE,

ETHANOL,

ETHANOL,

ETHANOL,

IN SEA-

24 HRS.

70%

90%

ABS.

WATER

XYLENE

FIG. 12. Graph showing how the volume of the
eggs of Arbacia pustulosa is affected by the addition
of non-fixative salts to formaldehyde solution, at
various stages in paraffin embedding. Each point
represents the mean of 10-15 measurements.

(From the data of Hertwig.***)

mammalian cells can be obtained by means of the system of grading
described on pp. 72 to 75. It will be remembered that the grad-
ing is done while the judge does not know what fixative was used
in the preparation of the slide: his judgement is therefore impartial.
The results with several fixatives are shown in table 7. In no case
was a fixative placed in a higher grade when used without an
indifferent salt than when used with it. The first three fixatives
in the table were markedly improved by the addition of an

REACTIONS OF FIXATIVES WITH TISSUES AND CELLS 83

TABLE 7

Grading of simple fixatives with and without the addition of indifferent
salts. ^^ The fixatives were used at the concentrations shown on p. 24.

Grade

Grade

Indifferent

Concentration

Fixative

zoithoiit

with

salt

of

iiidifferent

indifferent

used

indifferetit

salt

salt

salt, %

chloroplatinic

IV

II

sodium

075

acid

chloride

chromium

III

II

sodium

07

trioxide

chloride

formaldehyde

IV-V

III

calcium
chloride
(anh.)

i-o

picric acid

IV-V, V

IV

sodium
chloride

0-7

mercuric

IV

III-IV

sodium

06

chloride

chloride

osmium

IV-V, V

III-IV, III-

sodium

0-7

tetroxide

IV, IV

chloride

potassium

V

IV-V

sodium

0-7

dichromate

chloride

trichloracetic

III-IV

III-IV

sodium

0-7

acid

chloride

acetic acid

III

III

sodium
chloride

0-7

indifferent salt, and most of the others sHghtly ; with trichloracetic
and acetic acids, however, there was no difference.

The facts, then, are clear enough. Indifferent salts do improve
fixation by certain fixatives, especially the fixation of the cells of
marine algae and marine invertebrates. The reason for this is not
obvious. More than half a century ago the Swedish investigator
Sjobring *^* laid it down that formaldehyde should be used at such
a concentration as to make the fluid isotonic with the tissues to be
fixed. For mammalian tissues he used formaldehyde at 8 to 10%.
He should actually have used it at about 4% to achieve his pur-
pose,^^^ but the question is whether his principle was correct. The
evidence against it is conclusive. ^^^' ^^^' ^^ Fixatives may be made up
so as to be hypotonic, isotonic, or hypertonic to the body-fluids:
the results show that there is no virtue in isotonicity.^^^ Acetic
acid at 5% swells tissues, yet it exerts an osmotic pressure of 20
atmospheres, about three times that exerted by mammalian blood.
Chromium trioxide at ^%, with the very small osmotic pressure
of 1-4 atmosphere, shrinks cells strongly. Similar examples could
be multiplied. 2^ It is clear that the total osmotic pressure exerted

84 FIXATION

by the constituents of a fixative is unrelated to the sweUing or
shrinkage of cells.

According to several authors,^"' *^^' ^^*' ^^^ fixative substances
should be dissolved in a solution of an indifferent salt giving an
osmotic pressure equal to that of the intercellular fluids of the
tissues that are to be fixed. It follows that the fixative solution as a
whole should be hypertonic to these fluids.

It is claimed that when a piece of tissue is placed in a solution of
a fixative substance dissolved in distilled water, the cells in the
centre of the piece are affected as they w^ould be if it had been
placed in distilled water. The ions of the intercellular fluid diffuse
rapidly outwards into the fixative solution, while the fixative sub-
stance penetrates more slowly inwards. The intercellular fluid
therefore becomes hypotonic to the cells, and the latter accord-
ingly swell and burst. If the fixative is dissolved in a suitable saline
medium, the mobile ions of the latter counteract this process by
diffusing into the tissues ahead of the fixative substance. ^^^ It is
to be noticed that this theory postulates the presence of an inter-
cellular fluid. Now we have already seen that echinoid eggs swell
strongly when placed in a formaldehyde solution devoid of in-
different salts, but scarcely at all when this fixative is dissolved in
sea- water. Thus the initial swelling, postulated as the prime cause
of the damage, occurs also when there is no intercellular fluid.
Beyond this, it is to be held in mind that no one has proved that
cells do in fact burst when placed in fixative solutions lacking
indifferent salts. They appear shrunken in the final preparation. It
is not clear why a burst cell must shrink.

It is argued that fixative substances can pass easily through cell-
membranes, and that they are therefore not able to exert osmotic
pressure on the cell-contents. ^^^ It has also been argued that the
cell-surface is so much altered by fixation that it is no longer able
to act as an osmotic membrane when a fixative has reached it.^^^
Hertwig's experiments with echinoid eggs do not appear to support
these opinions. Formaldehyde comes up against the cell- membrane
directly the eggs are put in the solution, yet there is swelling unless
the fixative was dissolved in sea- water. This suggests that the cell-
surface continues to act as an osmotic membrane.

If the indifferent salt acts osmotically, it must perform its
function while remaining outside the cells. We have no proof that
in fact it remains outside. On the contrary, it is conceivable that
it enters the cells with the fixative and then produces the observed

REACTIONS OF FIXATIVES WITH TISSUES AND CELLS 85

results. Perhaps the salt acts on the proteins of the cell in such a
way as to affect the imbibition of water. Some protein gels have a
tendency to swell in the presence of non-fixative salts, in much the
same way (though not so much) as they swell in the presence of
acetic acid; this would counteract the shrinking effect of certain
fixatives. It is relevant to remark that when acetic acid is included
in a fixative, the presence or absence of an indifferent salt has
little effect. ^^^ The fact must be kept in mind, however, that
echinoid eggs swell in the absence of an indifferent salt.

If an indifferent salt acts within the cell, it does not necessarily
do so by affecting imbibition. Its influence may be on the reaction
of the fixative substance with the proteins of the cell. We have
already seen how the action of mercuric chloride on proteins is
affected by the presence of sodium chloride (p. 54). A still more
striking instance is provided by ferric sulphate, w^hich is rather a
useful fixative. ^^ By itself, a J% solution of the anhydrous salt
does not coagulate egg- albumin; in the presence of ammonium
sulphate it coagulates it instantly (see Appendix, p. 316). Ammo-
nium sulphate alone is devoid of the ability to coagulate egg-
albumin at any concentration, and it is therefore an indifferent
salt.

If indifferent salts exert their effects after entering cells with the
fixative substance, it does not follow that their osmotic pressure is a
matter of indifference. Such a salt may diffuse rapidly into the piece
of tissue and act osmotically, in the way suggested, until the fixative
substance, diffusing more slowly, reaches the cells in the interior;
it may then enter the cell with the fixative when the latter has
damaged the cell-surface and stopped it from acting as a semi-
permeable membrane.

It follows from what has been said that the use of indifferent salts
is reasonable, though acetic acid may in some cases replace them ;
that they should not be used indiscriminately, without considera-
tion of their possible effect on the coagulative powers of fixative
substances; and that they should be present at such a concentra-
tion that they exert about the same osmotic pressure as the inter-
cellular fluids of the tissues.

It has not been proved that the osmotic pressure of the indifferent
salts should be exactly the same as that of the body-fluids. A
slightly lower concentration seems to give better results. Thus
sodium chloride works well at 0-7% with the tissues of mammals,
and calcium chloride at 1%.^^

86 FIXATION

When living cells are put in hypotonic solutions, those that are
dividing have a tendency to swell strongly, and mitosis often comes
to a halt at prometaphase or metaphase; the chromosomes are
widely dispersed. ^^^ Cells may be fixed in this condition, which is
especially favourable for the counting of chromosomes.^^^'^^^'^^^'
An aqueous solution of sodium chloride at 0-3 to 0-5% is suitable
for mammalian cells. ^^''

There is no evidence that it is ever advantageous to dissolve
fixative substances in complex physiological saline solutions.

Urea ^ and saponine ^^^ are occasionally added to fixative solu-
tions. Their effects, if any, have not been critically analysed. There
is no concrete evidence that the reduction of surface tension is
helpful.

It is desirable that the whole subject of non-fixative substances
in fixative solutions should be reinvestigated. Some of the most
important problems remain unanswered. In the absence of in-
different salts, do cells in the interior of pieces of tissue actually
swell, burst, and then shrink? Do the indifferent salts exert their
effects outside or inside the cell, or first outside and then inside?
We simply do not know.

In the early days of microtechnique it was important that tissues
should be hardened so that they could be sectioned easily by hand.
With the introduction of effective embedding media, hardening
became less important. Nevertheless, the physical properties of
fixed tissues remained and still remain important. It is necessary
that the fixative should give them such a consistency that when
they have been embedded, they can be cut into sections easily.
They must not be friable, brittle, or very hard. Much depends on
the process of embedding. Some tissues, such as the lens of the
eye or thick pieces of voluntary muscle, are difficult to section in
parafiin but easy in collodion. In such cases the process of em-
bedding makes more difference than fixation.

It would be useful to have quantitative information about the
physical properties of various tissues at all stages of microtechnique
up to embedding. Unfortunately the available data are meagre.
Wetzel ^^^ studied the elasticity of the belly-muscle of the cat. He
cut out a long piece and put it in a fixative solution. He then
attached it by one end in such a way that it was held horizontally.
A weight attached to the free end caused it to sag. The coefficient
or modulus of elasticity was calculated from a formula involving

REACTIONS OF FIXATIVES WITH TISSUES AND CELLS 87

the dimensions of the piece of tissue, the weight appHed at the free
end, and the amount of sag. The higher the coefficient, the more
rigid the tissue. Unfortunately he made only one or two obser\^a-

5000

4000-

t 3000
u

<

o

2000

o

1000

ACETONE

ABS. ETHANOL,
ABS.

o

>-

X

<

a.
o

<

o

_l

I
u

o

u
a:

X

o

o

I
u

I

O
X

o

2
2

<
2
o

I
u

o

2

10

<

<
to

<

■ ^ J1

<

<

FIG. 13. Diagram showing the coefficient of elasticity of the belly-
muscle of the cat, fixed in various ways. The black columns show
the coefficient after fixation, the white columns after fixation and
subsequent soaking for 4 days in 8o°o ethanol.
(From the data of Wetzel. "=)

tions with each fixative. Sometimes he transferred the tissue from
the fixative to 80% ethanol and measured the elasticity once more.
Some of his results are reproduced graphically in fig. 13. The
excessive hardening effect of acetone and ethanol will be noticed.
No other fixative approaches these two. Potassium dichromate,

88

FIXATION

picric acid, and acetic acid leave the tissue very soft and incapable
of being hardened to any considerable extent by subsequent
soaking in 80% ethanol.

The effect of fixation on dyeing will be considered later in the
book (p. 202), when the nature of dyes and the way in which they
attach themselves to tissue-constituents have been discussed.

CHAPTER 5

Primaij Fixatives Considered
Separately 1 . Coagulants

The term 'primary fixatives' is used here to mean single fixative
substances as opposed to mixtures of tvvo or more in a single solu-
tion. Primary fixatives may be used either absolutely, as ethanol,
for instance, is sometimes used; or in solution in distilled water;
or in solution in water with an indifferent salt.

The action of fixative mixtures can only be understood through
a process of analysis. It is for this reason that the preceding
chapters have been concerned almost entirely with primary fixa-
tives. In this chapter and the next such fixatives will be discussed
one by one. The various facts that have already been related about
them will here be brought together in summary form, and further
information will be added so as to afford as many-sided a view of
each as possible. Certain important facts about fixation are not
well suited to the kind of general treatment adopted in the earlier
chapters, but lend themselves to separate exposition under the
heads of particular fixatives.

It is thought that the principles of fixation can be explained
better by a fairly full account of a few primary fixatives than by a
sketchy one of many. The eight described in these two chapters
have been chosen partly because they are components of familiar
mixtures, partly because they are diverse in action and illustrate
many important points about fixation. The selected primary
fixatives are these: —

coagulant

ethanol. .... This chapter, p. 92

picric acid .... ,, p- 96

mercuric chloride ... ,, P- 99

chromium trioxide . . „ p. 104

89

QO FIXATION

non-coagulant

formaldehyde . . . Chapter 6, p. 1 1 1

osmium tetroxide . . . ,, p. ii8

potassium dichromate . . ,, p. 126

acetic acid .... ,, p. 134

These fixatives will be considered one by one under a standard-
ized set of headings. The headings are listed below, with short
comments w^here necessary.

Standard concentration for fixation. Most of the simple fixatives
can be used over a fairly wide range of concentrations, but it is a
great convenience to select a rather arbitrary 'standard' concentra-
tion, somewhere within this range. It is better to know something
about 5% acetic acid, for instance, from all points of view, than to
have disjointed information about its pH at i %, its rate of penetra-
tion at 2'5%, and its swelling effect at 10%. The standardization
of concentrations also makes it possible to avoid unnecessary
repetition. Wherever in this book a primary fixative is mentioned,
it is to be understood, unless the contrary is distinctly stated,
that the information refers to its use at the standard concentration,
or at a concentration so close to this that the difference is
insignificant (see pp. 24 and 32).

Description. Information about the appearance of each sub-
stance and its melting- and/or boiling-point, solubility, etc., is
collected under this heading. Industrial uses are also mentioned.

Ionization. The figures given in the literature for the pH of
aqueous solutions at the concentrations suitable for fixation are
not always in good accord. This must be attributed partly to the
impurity of the chemicals commonly used in biological labora-
tories. Two of the fixatives are used at saturation, and temperature
may have a considerable effect on the concentration of such
solutions and hence on pH; it may also affect the pH of other
fluids.

The data on change of pH during fixation are those of Freeman
and his collaborators ^"^ and Casselman.^^^ Freeman generally
allowed i g of spinal cord or liver of cat to 25 ml of fixative
solution.

Oxidation-poteiitial. The oxidation-potentials of fixatives have
been investigated by Casselman,^^^ whose figures are quoted in
this chapter.

Manufacture. For fuller information about proce$§Q§ of manu-

PRIMARY fixatives: COAGULANTS 91

facture than is given in these chapters, see especially Thorpe and
Whiteley ^o* and Kirk and Othmer.^e?

Introduction as fixative. Care has been taken to provide accurate
information on this subject. Historical matters are often handled
carelessly in scientific works.

Reactions with proteins. Readers who want further information
on this subject, beyond the summaries given here, should turn
back to chapters 2 and 3. In the present chapter and the next,
brief notes are given under this heading about the effects of fixa-
tives on solutions of nucleic acids, because this follows naturally
on the discussion of their effects on nucleoproteins.

Reactions with lipids and Reactions with carbohydrates. The
information under these headings is not given in any other part of
the book.

Rate of penetration. The remarks made on this subject in these
chapters summarize what has already been said about it in chapters
2 and 4; some additional facts are added. The K-values given are
based on the following data: —

25 hours' penetration into gelatine/albumin gel; ^^
12 hours' penetration into mammalian liver; ^°°
25 hours' penetration into liver of rabbit. ^^

It follows from what has been said in earlier chapters that the
K-values would not have differed much if other periods had been
chosen.

It is important for the reader to remember that fixatives pene-
trate much faster into gelatine/albumin gel than into liver. Thus
a K-value of o-8 is very slow for penetration into the gel, but
moderate for penetration into liver.

Shrinkage or swelling. The figures for the shrinkage or swelling
of gelatine/albumin gels represent the volumes after 18 hours'
fixation. The results obtained by different authors with different
organs and cells are not entirely concordant. The attempt is made
to present a general picture of the effect of each fixative in causing
change of volume and in leaving the tissues subject to such change
on subsequent treatment. For further information on this subject,
see pp. 36 and 75.

Hardening. There is unfortunately no quantitative information
beyond the rather meagre data of Wetzel ^^^ (see p. 86).

Immediate effects on particular constituents of the cell. Under this
heading an attempt is made to integrate the results obtained with

92 FIXATION

various cells by several authors, especially Strangew^ays and
Canti ^^^ and Policard, Bessis, and Bricka.*^^ The effects described
are those produced by the fixative itself, without subsequent
dehydration or other treatment.

Methods of washing out. No comment is necessary here.

Effects on dyeing. The effects of different fixatives on the affinity
of tissue-constituents for dyes have only been briefly touched on in
the preceding part of the book, because it is thought better to dis-
cuss this subject in the part dealing w^ith dyes (p. 202). The in-
formation is nevertheless summarized here, so that no one who
uses chapters 5 and 6 for reference will be inconvenienced by
the absence of any mention of this important aspect of fixation.

Effects on the histological picture seen in parajfin sections. The
grading of fixatives, mentioned under this heading, has been fully
described on pp. 72 to 75. It is important to remember that this
system of grading is based on judgements made from the point of
view of routine histology. The fact that a fixative falls in one of the
lower grades by no means necessarily condemns it. It may have
virtues that are only brought out by mixture with other fixatives,
or it may be useful for some particular purpose in cytology or
histochemistry, or give good results when some other method of
embedding is used.

The attempt is made under this heading to present a general
picture of what is seen in the finished preparation, by integrating
the results of the grading- test with the findings of Tellyesniczky,^^'
Zirkle,^^^' ^" Pischinger,*^^ and Casselman.^^^ These authors did
not all use the various fixatives at exactly the same concentra-
tions ; but despite this, and despite the diversity of the cells stud-
ied, it is possible to draw certain general conclusions.

Compatibility with other fixatives . No comment is necessary here.

ETHANOL (ethyl alcohol)

Standard concentration for fixation. Absolute (100%).

Formula and formula-weight. C2H5OH. 46-0.

Description. Ethanol is a light fluid (specific gravity 0-791 at
20° C), miscible with water in all proportions. It boils at 78° C and
solidifies at about — 112° C. If free from water it gives no cloudi-

PRIMARY FIXATIVES: COAGULANTS 93

ness on mixture with benzene and does not produce gas (acetylene)
on contact with calcium carbide. Ethanol is a powerful dehydrating
agent.

Ionization. Not ionized.

Oxidation-potential. Ethanol at 95% shows an oxidation-
potential of 0*45 volt.^^^ Of the eight fixatives considered in this
chapter and the next, only formaldehyde shows a lower potential.

Manufacture. It is usually prepared from malt or molasses by the
action of yeast, with subsequent distillation. Fractional distillation
does not give a higher concentration than 95%. Distillation of this
strong ethanol at 81° C in the presence of anhydrous calcium
sulphate or some other suitable dehydrating agent will give
ethanol at 99-95%.

Ethanol is azeotropic with benzene: that is to say, it forms wdth
benzene a binary mixture W'ith sharp boiling-point (68° C). It also
forms a ternary azeotropic mixture with benzene and water, with
even lower boiling-point (65° C). If benzene is added to ethanol
containing some water and the mixture is distilled, the ternary
mixture comes off first, then the binary: nearly pure ethanol is left
behind, slightly contaminated with benzene. The latter can be
removed by filtering through active charcoal. It is called 'azeo-
tropic' ethanol, though actually it consists of that fraction of the
ethanol that did not form an azeotropic mixture.

Introduction as fixative. Wine was used in embalming by the
ancient Egyptians, but not as the principal preservative. Ethanol
appears to have been first used as a preservative for specifically
anatomical purposes by Robert Boyle at Oxford in 1663.^^^ The
use of spirits of wine for the preservation of animals was well
established before the middle of the eighteenth century. It was the
examination of a spirit-specimen of Alcyonium, collected by
Bernard de Jussieu on the coast of Normandy, that convinced
Reaumur that this and other zoophytes were in fact animals, ^^^
and Reaumur himself *^* subsequently recommended the use of
the same fluid for the preservation of birds. Henry Baker ^^ preserved
hydra in spirits of wine, but subsequently allowed his specimens
to dry. The special virtue of ethanol when mixed with acetic acid
was discovered by Clarke ^^^ in 185 1.

Reactions with proteins. Ethanol is a non-additive, coagulant
fixative. Coagulates are digestible by pepsin and typsin. In denatur-
ing proteins it shifts their iso-electric points less than other fixatives
do. Its capacity to denature is much affected by temperature: there

94 FIXATION

is no measurable denaturation below — 15° C. It slowly gelatinizes
histones.^^^ It does not denature zein or gliadin, nor does it stabil-
ize gelatine gel against solution by warm water. It converts
haemoglobin into kathaemoglobin ; ^^^ that is to say, it denatures
the globin without splitting off the haem.

Ethanol is a non-coagulant of nucleoprotein. Nucleic acids are
precipitated from solution, ^^^'^^^ but not rendered insoluble in
water.

Reactions with lipids. Ethanol does not fix lipids in the sense of
rendering them insoluble in lipid-solvents.

Tripalmitin and tristearin are almost insoluble in cold ethanol,
triolein somewhat soluble; similarly palmitic and (especially)
stearic acid have only slight solubility in the cold fluid, while oleic
acid is readily soluble. Cholesterol is slightly soluble, the common
cholesteryl esters nearly insoluble. Lecithin is soluble ; the ethanol-
amine and serine esters of phosphatidic acid, together constituting
'cephalin', are insoluble, except in the presence of lecithin.
Sphingomyelin and the cerebrosides are insoluble. ^^^' ^^ Plasmalo-
gen is soluble in ethanol, but when associated with elastic fibres
it is not extracted by this solvent. ^^^

Thus among the common lipids, only triolein, oleic acid,
lecithin, and plasmalogen show more than slight solubility in cold
ethanol. Most lipids are soluble in hot ethanol, but this is not used
for fixation.

Dilute ethanol is an 'unmasking' agent, capable of splitting lipids
off from certain lipoprotein complexes. ^^^

Reactions with carbohydrates. Ethanol does not fix carbo-
hydrates. Glycogen is insoluble in it and is therefore precipitated
unchanged.

Rate of penetration. Ethanol penetrates slowly into clotted
blood-plasma,^^^ but it goes rather quickly into tissues. ^^^' ^^^' ^^^
Tellyesniczky's ^^^ and Seki's ^^^ results with mammalian liver
agree closely. The K-value obtainable from their data is almost
exactly i-o.

Shrinkage or swelling. Ethanol shrinks gelatine/albumin gel
more strongly than any other fixative except acetone. ^^ It also
shrinks whole livers strongly, though not so much as chromium
trioxide and picric acid do.^^ It shrinks Arbacia eggs to less than
half their original volume. The primary spermatocytes of Helix
aspersa are shrunk to 19% of their original volume when paraffin
sections of ovotestes fixed in ethanol have been mounted in

PRIMARY fixatives: COAGULANTS 95

Canada balsam. No other fixative studied by Ross ^^^ gave so
much shrinkage of cells in final preparations. There is evidence
that nuclei are not so badly shrunk as whole cells. ^^^

Hardening. Ethanol hardens tissues extremely, more so than any
other fixative except acetone. Wetzel's ^^^ figure representing
rigidity (elasticity) is about 4600 (about 20 times as great as the
figure for chromium trioxide).

Immediate effects on particular constituents of the cell, A coarse
coagulum is produced throughout the cytoplasm; mitochondria
are destroyed; lipid globules tend to fuse and may dissolve. A
coarse coagulum appears also in the nucleus; the nucleolus is
shrunken.

Methods of washing out. Ethanol is miscible in all proportions
with toluene and similar antemedia for paraffin embedding, and
also with ether (and with water). It follows that no special washing
out is necessary.

Effect on dyeing. Ethanol has less eflPect on the dyeing of proteins
than most fixatives have. Albumin remains readily colourable by
both basic and acid dyes: protamine, strongly basic before fixation,
remains acidophil afterwards. ^^^ Fixed cytoplasm reacts to dyes
like fixed albumin; chromatin is rendered strongly colourable by
basic dyes, but its distribution in the cell is not stabilized, and
ethanol is therefore a very poor fixative for chromosomes at all
stages.

Effects on the histological picture seen in paraffin sections. By
itself, ethanol is a poor fixative (grade IV). Cellular aggregates tend
to shrink apart from one another, leaving rather large spaces in
between; cells also tend to separate from one another; cytoplasm
contracts strongly. Red blood-corpuscles are not badly fixed,
partly because haemoglobin is preserved (as kathaemoglobin ^^^),
partly because the chief distortion to w^hich they are subject is
swelling, and ethanol prevents this. Nuclei are fixed without much
distortion of shape; a nuclear network is not produced. Chromo-
somes are not distinctly seen.

The cytoplasm often contracts in such a way that it is piled up
against the cell-membrane on the side opposite to that from
which the fixative penetrated. Any glycogen present in the cell
will be found in the displaced mass of cytoplasm. The contents
of the nucleus are often piled up against the nuclear membrane
in the same way. This curious orientation of the protoplasm has
not been satisfactorily explained. It is presumably connected with

g6 FIXATION

the considerable reduction in volume that takes place when
ethanol is mixed with water.

Compatibility with other fixatives. Ethanol is compatible with
picric acid, mercuric chloride, formaldehyde, and acetic acid, but
it reacts with the latter substance to produce ethyl acetate if
sulphuric acid be added to the mixture. Since ethanol tends to be
oxidized through aldehyde to acetic acid, it is best not to mix it
with chromium trioxide, potassium dichromate, or osmium
tetroxide. It does not react quickly with potassium dichromate,
however, unless the mixture be acidified. The reaction with
osmium tetroxide to produce a black precipitate is also slow at
room-temperature.

Unclassified remarks. So far as is known, ethanol is the only
non-aqueous fluid, other than methanol and acetone, that gives
reasonably good results when used undiluted as a fixative, ^^^
though some might make a claim for dioxane.

PICRIC ACID (trinitrophenol)

Standard concentration for fixation. Saturated aqueous solution.

OH

Formula and formula-weight. ^11 ^. 229-1

NO2

Description. Picric acid consists of yellow leaflets or prisms,
melting at 122° C. It is slightly soluble in water (1-4% w/w), more
so in ethanol (4-9%), stiU more in benzene (10%). Its solubility
in ethanol and ether often results in the yellow coloration of col-
lodion blocks containing tissues fixed in picric acid.

Picric acid decomposes explosively when heated suddenly or
detonated, and is therefore usually damped for storage. Until
displaced by the related trinitrotoluene, it was the chief high
explosive used for military purposes. It was manufactured at
Lydd in Kent and received the name of lyddite. At the present
day it is still used as an explosive in the form of its potassium,
sodium, and ammonium salts.

This is the only substance that is used in microtechnique both
as a fixative and as a dye (p. 185).

The name is derived from the Greek work meaning bitter.

Ionization. This is a much stronger acid than phenol. The pH of
the saturated solution has been given as 1-33 ^^^ and i-6.*^^

PRIMARY fixatives: COAGULANTS 97

Oxidation-potential. The saturated solution shows an oxidation-
potential of 0-82 volt/^^ a little more than that shown by a 5%
solution of mercuric chloride.

Ma?mfacture. Picric acid may be prepared by the direct action
of nitric acid on phenol, but this is wasteful because part of the
phenol is lost by oxidation. In industry the ortho- and para-
sulphonic acids are first formed, by treating melted phenol with
concentrated sulphuric acid. The resultant phenolsulphonic acids
are dissolved in water and treated with nitric acid; NO 2 groups
now occupy the ortho- and para-positions.

Introduction as fixative. Picric acid was mentioned as a hardening
agent by Ranvier ^^^ in 1875, but it is not known who first intro-
duced it into microtechnique. It was much used by Flemming ^"^' ^^^
from 1879 onwards in his pioneer w^ork on chromosomes.

Reactions with proteins. Picric acid is a coagulant fixative. It is a
particularly strong coagulant of the histone of the nucleus. ^^^ It
does not stabilize gelatine gels. It forms crystalline compounds
with amino-acids and is probably to be regarded as an additive
fixative of proteins, though its precise points of attachment have
not been disclosed. It is an alkaloidal reagent, and the claim is
made that it fixes basic proteins while allowing acidic ones to
escape by solution. *^^ Coagulated protein is digestible by pepsin
and trypsin.

With nucleoprotein solution picric acid produces fine flocculi,
which are eventually precipitated. DNA is either left in solution,
or precipitated ^^^ in a water-soluble form.^^^ The fact that picric
acid may precipitate the protein of nucleoprotein while leaving
nucleic acid in solution has been used in the preparation of DNA.^^^

Reactions with lipids. None has been recorded. Lipids are not
dissolved by aqueous solutions of picric acid.

Reactions zvith carbohydrates. Picric acid is not a fixative of
carbohydrates, but is strongly recommended by Lison ^^^ as a
constituent of fixatives for glycogen. The latter substance is
apparently bound to protein (sometimes quite firmly as 'desmo-
glycogen'), and picric acid not only does not set it free, but acts
on the protein in such a way as to cause the glycogen to resist
solution.

Rate of penetration. Picric acid penetrates into gelatine/albumin
gel more slowly than any other fixative that has been tried (K =
0-8). It also goes rather slowly into liver (K = 0-5) ^^ (see fig. 5, A,
opposite p. 67). Tellyesniczky ^^^ and Underbill ^^^ i^Q^h tested

G

98 FIXATION

it at about half- saturation and found slow penetration into liver
(K = o-4).5oo

Shrinkage or swelling. Picric acid has little effect on the volume
of gelatine/albumin gel, but it shrinks whole livers to 74% of
the original volume; after infiltration with paraffin the volume is
reduced to 42%. This represents greater shrinkage at the paraffin
stage than that produced by the other fixatives studied by Berg.^^
It is evident that the shrinking effect of coagulation outweighs any
tendency to swell as a result of acidity. The total shrinkage of the
primary spermatocyte of Helix aspersa caused by fixation in picric
acid and subsequent treatment up to the mounting of paraffin
sections in Canada balsam, is excessive: the cell only occupies 20%
of its original volume. ^^^ Ethanol is the only fixative that produces
greater final shrinkage than this.

Hardening. It is strange that this fixative, which rivals ethanol
in the shrinkage it produces, comes almost at the opposite end of
the scale as a hardener. Ethanol hardens tissues excessively: picric
acid leaves them very soft, and incapable of being much hardened
by 80% alcohol. Wetzel's figure for rigidity is 69. That for chro-
mium trioxide is about 3-4 times as great. The value of picric acid
as a fixative lies partly in the soft consistency it gives to tissues.

Immediate effects 07i particular constituents of the cell. Pseudopodia
tend to be constricted into separate globules; the ground cyto-
plasm is coagulated with varying degrees of coarseness; mito-
chondria are not destroyed, but sometimes become moniliform;
lipid droplets may fuse. The nuclear sap is coagulated, the
nucleolus somewhat shrunken. Chromosomes are rather well
preserved.

Methods of washing out. It is sometimes said that protein
coagulated by picric acid is soluble in water, and that care should
therefore be taken to transfer tissues directly to ethanol. This
does not appear to be true, but nevertheless there is generally no
advantage in washing out in water, since the excess of fixative is
more readily removed by ethanol or indeed by benzene or other
antemedium used in paraffin embedding. Any yellow colour
remaining in sections may be removed by the action of an aqueous
solution of lithium carbonate,^^^ or replaced by the action of some
other acid dye.

Effect on dyeing. Picric acid renders egg-white acidophil. The
effect on cytoplasm is similar: very little affinity for basic dyes is
retained. When nucleoprotein is coagulated by picric acid, the

PRIMARY fixatives: COAGULANTS 99

DNA remains in solution (compare Levene -^^). Something similar
appears to occur in the fixation of tissues, for chromatin fixed by
picric acid has little affinity for basic dyes, while some forms of it
are rendered quite strongly acidophil. ^^ It must be supposed that
in these cases a basic protein is separated from DNA and coagu-
lated, while the DNA dissolves.

Ejfects on the histological picture seen in parajfin sections. Even
with the help of an indifTerent salt, picric acid by itself is a grade
IV fixative. There is little tendency for cellular aggregates to
shrink away from one another, but cytoplasm generally assumes
a very coarsely reticular form, or contracts into a mass surrounding
the nucleus, or even disintegrates. Red blood corpuscles swell and
become spherical. The shape of nuclei is rather well preserved;
the nuclear membrane is clearly shown; a nuclear network is
produced.

Compatibility with other fixatives. Picric acid is very tolerant of
mixture with other fixatives. It may be used with any of the others
described in this chapter and the next.

MERCURIC CHLORIDE

Standard concentration for fixation. Saturated aqueous solution.

Formula and formida-weight. HgCU. 271-5.

Description. The crystals are long, white needles. The molecule
has a linear shape, the chlorine atoms being at opposite poles of
the mercury atom. The melting-point is 275^ C, but the boiling-
point is not far above (301 ') and the substance sublimes easily. At
room-temperatures mercuric chloride dissolves in water at from
6-6 to 7*1% w/w. It is also readily soluble in ethanol and benzene.

This substance is used as an antiseptic and disinfectant, in the
dressing of furs, in the intensification of photographic negatives,
and in the preparation of various mercuric compounds.

Since mercuric chloride is usually prepared by sublimation
and in strong solution is corrosive to the tissues of the mouth, it
used to be called corrosive sublimate. When taken into the body
in small quantities it may cause acute nephritis, the tubules and
sometimes the glomeruli being affected; the formation of urine is
reduced and may cease, with consequent death.

Ionization. The electrical conductivity of solutions of mercuric
chloride is low, because of limited ionization. Solutions of this
substance are extremely complex. Partial hydrolysis is said to

100 FIXATION

give (HgCl)20 or HgClOH, with hydronium and chloride ions.
[HgClJ^, [HgCl]^, and the mercuric [Hg]^^ ion are present, in
addition to undissociated HgClg.^^' [HgClJ^ is particularly
readily formed in the presence of other chlorides, and mercuric
chloride is more soluble in solutions of sodium chloride than in
distilled water (about io% in 0-75% sodium chloride).

The hydronium ion produced by hydrolysis results in moderate
acidity. The figures given for the pH of saturated aqueous solu-
tions vary from 2-8 to ^.6.i^6'285, 397, 459 j^ careful reading of the
pH of a 5% solution gives 3-25.^^^

Solutions of mercuric chloride become more acid during fixa-
tion. ^^^ All other acidic fixatives maintain their pH or become less
acid during fixation.

Oxidation-potential. The mercuric ion is capable of reduction to
the mercurous [Hga]^^ ion, and mercuric chloride can therefore
act as an oxidizer. A 5% aqueous solution shows an oxidation-
potential of 0-75 volt. This is a little less than the figure for 2-5%
potassium dichromate and a little more than that for 2% osmium
tetroxide. Thus mercuric chloride is a moderately strong oxidizer.

Manufacture. Metallic mercury is dissolved in concentrated
sulphuric acid; the solution is heated and sodium chloride is
added, with a little manganese dioxide. Mercuric chloride is
formed. The temperature is increased and it sublimes. It is caught
on the interior surfaces of glass funnels.

Alternatively mercuric chloride may be made by the direct
chlorination of mercury. The metal 'burns' in an atmosphere of
chlorine. The product sublimes and is deposited on the walls of
the chamber in which the combination occurs.

Introduction as fixative. Mercuric chloride appears to have been
first used as a preservative for anatomical specimens by the French
surgeon and anatomist, Chaussier (1746-1828).^^ Goadby included
it in his preservative mixture, which was recommended by
Quekett; *^^ but the substance was so dilute (about 0-023%) "^^^^
it cannot have acted as a fixative, and Goadby's fluid must have
relied upon its other constituents for its preservative qualities. In
1847 the distinguished French morphologist Blanchard '^^ added
a solution of mercuric chloride to sea- water to fix marine Turbel-
laria. Corti ^^^ used it as a fixative in his histological study of the
inner ear, and so did Remak *^^ in his work on the multinucleate
cells of the liver.

Lang, the celebrated Swiss zoologist and microtechnician.

PRIMARY fixatives: COAGULANTS lOI

alighted by chance on Blanchard's paper and decided to try mer-
curic chloride as a fixative, first ^^^ with marine Turbellaria in
1878, afterwards -^-^ with a wide variety of marine invertebrates.
He used it both as a simple saturated solution and also in mixtures.
The popularization of mercuric chloride as a fixative was due to
Lang. Unfortunately he gave no exact reference to Blanchard's
paper, and Whitman, ^^" in his well-known textbook of microtech-
nique, gave a wrong one. These facts have clouded Blanchard's
priority of 31 years. Ranvier ^^- mentioned mercuric chloride in
1875 ^s a substance used in histological technique, but did not say »
for what purpose.

Reactions with proteins. This is a powerful coagulant of soluble
proteins. The coagulate is not so readily digestible by pepsin or
trypsin as that produced by ethanol or picric acid. Histone is
gelatinized.^^- Gelatine gels are not stabilized.

Mercuric chloride reacts with the sulphydryl groups of the
cysteine component of proteins, forming links between protein
chains; but in the circumstances of ordinary fixation much more
mercury is taken up in other ways. On the acid side of the iso-
electric point of the protein it is taken up chiefly by amino-
groups, as [HgCl4]". The combination is very loose; it is promoted
by the presence of added chloride (for instance, sodium chloride).
On the less acid side of the iso-electric point the molecule is taken
up as a whole, as HgClg. It associates with amino-groups, by
which it is loosely held; the combination is opposed by chloride
and by iodide. These reactions with amino-groups are coagulative.
It is perhaps relevant that mercuric chloride is said to be less
antiseptic in the presence of sodium chloride. ^"^ In alkaline solu-
tion mercuric ions are taken up by carboxyl groups in the protein.

Mercuric chloride is not a very powerful coagulant of nucleo-
protein: fine fiocculi are produced. It is also a weak precipitant of
nucleic acids. ^^^' *^^

Reactions zvith lipids. Mercuric chloride is not known to react
with triglycerides. There is evidence,"^ however, that these colour
less strongly with Sudan III and IV (p. 299) and with Nile red
(p. 301) after treatment with this fixative than after treatment with
formaldehyde. Mercuric chloride forms compounds with phos-
pholipids,^^ but the solubilities of such compounds appear not to
have been investigated. The solubility of certain conjugated lipids
in lipid-solvents is supposed to be reduced by treatment with salts
of cadmium (compare Ciaccio '^^^), and since this metal is related

102 FIXATION

to mercury, we may perhaps expect that mercuric chloride will
be found to be a fixative for these lipids.

The salts of the three related metals, mercury, cadmium, and
zinc, are said to separate certain lipids from lipoprotein complexes
and thus 'unmask' them.^^*

The most striking property of mercuric chloride in relation to
lipids or lipid-like substances is its capacity to hydrolyse plasma-
logen in such a way as to separate plasmal (chiefly palmitic and

H2C— 0\ O^

I /CH(CH2)nCH3 /C(CH2)i,CH3

HC— 0/ O H^

T I!

H2C— O— P— CH0.CH2.NH2

OH

Plasynalogen Plasmal

Stearic aldehydes) from glyceryl-phosphoryl-ethanolamine. The
plasmal can then be made visible by the use of Schiff's aldehyde-
reagent (p. 308).-^^''' ■^^^' ^^^' ^^"" It is not fixed by mercuric chloride,
but retains its solubility in lipid-solvents (unless it chances to be
adsorbed on elastic fibres). ^^^

Reactions with carbohydrates. None is described. Mercuric
chloride is regarded as a particularly good fixative for certain muco-
polysaccharides called by histologists 'mucin'. ^^^

Rate of penetration. Mercuric chloride penetrates at moderate
speed into gelatine/albumin gel (K = 2-2). There is good agree-
ment between different investigators about the rate of penetration
into mammalian liver. The observations give K-values of 0-78 ^^^
and 0-84.^^ This also is moderate speed. For a photograph showing
the penetration of mercuric chloride into liver, see fig. 5, a (oppo-
site p. 67).

Shrinkage or swelling. Mercuric chloride only produces a
small reduction in the linear measurements of gelatine/albumin
gels and whole livers: the volume is reduced by less than 10% in
both cases. There is, however, further contraction to 70% of the
original volume on embedding liver in paraffin. Cells are much
more variable in volume after the action of this fixative than gels
or whole organs are. Arbacia eggs swell considerably in a saturated
aqueous solution of mercuric chloride, but by the time they are in
xylene they occupy only 49% of their original volume. Spermato-
cytes of Helix occupy 30% of their original volume when paraffin
sections have been mounted in Canada balsam. It must be re-

PRIMARY fixatives: COAGULANTS IO3

membered that there is more shrinkage than this after the action of
most fixatives (see fig. lo, p. 79).

Hardening. Mercuric chloride hardens moderately. Wetzel's
figure is about iioo, nearly 5 times that for chromium trioxide.
There is considerable further hardening on subsequent soaking in
80% ethanol.

Immediate effects on particular constituents of the cell. The shape
of the cell, including any pseudopodia, is rather well preserved.
The cytoplasm is sometimes much more finely coagulated than
one would expect of a fixative of this sort. Indeed, mercuric
chloride is particularly recommended by Policard and his col-
leagues *^* on this account. Cytoplasmic inclusions of various
sorts, including mitochondria and the neutrophil granules of poly-
morphs, are preserved, though lipid globules may fuse. The
nuclear membrane is clearly seen; nuclear sap is finely coagulated,
the nucleolus very distinct. The mitotic spindle appears fibrous.
Mercuric chloride distorts the cell less than any other coagulant
fixative.

Methods of washing out. Since mercuric chloride is readily
soluble in ethanol, there is no purpose in washing in water if the
tissue is going to be dehydrated.

Mercuric chloride has a troublesome tendency to produce ex-
trinsic artifacts. These appear in the final preparation as small,
black, amorphous particles, down to about i^ in diameter, and
also needle-shaped, birefringent crystals, up to 25/x long, usually
with a lump of the black material at each end. Both artifacts have a
tendency to be deposited eventually on the surfaces of the slide
and coverslip, in contact with the tissue.

Mayer ^^^ concluded that the black material was metallic mer-
cury, but he could not determine the chemical composition of the
crystals. It is unlikely that they consist of calomel, since they are
also seen after the use of Millon's reagent,^*^ which contains no
chloride. Mayer was unable to produce such crystals from mercuric
chloride or Millon's reagent except by their action on the tissues of
organisms. The crystals, unlike the black material, are not present
as such until the tissue is brought into a mounting medium, such
as Canada balsam; cedarwood oil is particularly apt to produce
them.

The black artifact is removed by the action of iodine in alcoholic
solution. This was discovered in 1886.^-^'^^^ Presumably the
mercury is oxidized to mercuric iodide, which is soluble in

104 FIXATION

ethanol. The precursor of the crystals is also removed by this
treatment.

The removal of mercury deposits may be delayed until sections
have been prepared, but it is best to treat the tissue in bulk w^ith
iodine solution, as this makes it less brittle and therefore easier to
cut. When sections have been prepared but not yet dyed, a glance
through the microscope will show w^hether a second treatment with
iodine is necessary.

Since protein coagulates may, in certain circumstances, be
soluble in iodide solutions (p. 54), it is best not to dissolve the
iodine with potassium iodide.

The colour of iodine is removable by soaking in 70% ethanol, or
almost instantly by the action of sodium thiosulphate. This method

2[S203]= + I2 = [S40e]= + 2I-

thiosulphate tetra- iodide

thionate

of removing iodine was introduced into microtechnique by
Heidenhain (1909).

Effect of dyeing. Within the range of pH at which it is ordinarily
used in fixation, mercuric chloride leaves the tissues more re-
ceptive of dyes in general than any other fixative. Cytoplasm will
accept both basic and acid dyes, particularly the former. Chromatin
is rendered very strongly colourable by basic dyes and dye-lakes.

Effects on the histological picture seen in paraffin sections. Mercuric
chloride by itself gives grade IV fixation. Cell- aggregates are not
seriously distorted. Cytoplasm is rather homogeneously fixed, but
it is badly shrunken and there is a tendency for cells to separate
from one another. Red blood-corpuscles are not badly fixed;
haemoglobin is retained within them (perhaps as methaemoglo-
bin *^^). The nuclear membrane is clearly shown; a nuclear net-
work is not produced; chromosomes are not well fixed.

Compatibility with other fixatives. Mercuric chloride may be
mixed with any other fixative. In certain mixtures, acetic acid
appears to reduce its solubility. ^^*

CHROMIUM TRIOXIDE

Standard concentration for fixation. 0-5% w/v aqueous solution.
Formula and formula-weight. CrOg. loo-o.
Description. Chromium trioxide consists of brownish-red crystals,
readily deliquescent by absorption of water. Their melting-point

PRIMARY fixatives: COAGULANTS

105

[HCr04-]

[Cr20,=]

hCrOj

[HCro/]

[Crp,-]

is about 198" C. They are extremely soluble in water, a saturated
solution having a concentration of 62-4% w/v.

Ionization. For a careful study of this subject, see Casselman.^^^

When chromium trioxide
is placed in water, it forms
chromic acid; this may be
regarded as H2Cr04, but no
such substance can be iso-
lated. The acid ionizes to
form hydronium ions and
three different chromic
anions. These are the orange-
red dichromate [CrgO^]"
and hydrogen chromate
[HCr04]^ ions, with traces
of the yellow chromate
[CrOJ" ion: a certain
amount of chromic acid re-
mains undissociated. The
relative proportions of the
four substances are shown in
fig. 14 (left-hand column).

The hydronium ions re-
leased by the ionization of
chromic acid make the solu-
tion strongly acid. A 1%
solution of analytical-grade
chromium trioxide has a pH
of 1-20.^^^ (Lassek ^^^ gives
I -12.) If chromium trioxide
of 'laboratory reagent grade'
is used, the pH of solutions
is significantly higher. ^^^

There is a very small rise
in pH (about o-i unit) dur-
ing fixation, almost entirely
in the first 3 or 4 hours. ^^^

Oxidation-potential. The dichromate ion can readily be reduced
to give the chromic ion Cr^~^. The oxidation-potential of a 1%
solution of chromium trioxide is i-o8 volt.^^^ Data do not exist for a
0-5% solution, but extrapolation from available figures ^^'^ makes

o

Otd
•_• —

E o
.5 E

{^ 2

H2Cr04] and
t [Cr04l

FIG. 14. Graphical representation
of the ions present in a 2-5%
aqueous solution of potassium
dichromate and in a solution of
chromium trioxide containing the
same weight of chromium.

(From Casselman/*- by kind permis-
sion of himself and of the Company of
Biologists, Ltd.)

I06 FIXATION

it unlikely that the potential would differ significantly from this.
Chromium trioxide is a much stronger oxidizer than any other
fixative.

The oxidation-potential does not change measurably even
during prolonged fixation. ^^^

Manufacture. Chromium trioxide is prepared by mixing excess
of concentrated sulphuric acid with a saturated aqueous solution
of potassium or sodium dichromate.

Introduction as fixative. Chromium trioxide was introduced into
microtechnique by Hannover in 1840. He had been staying in
Copenhagen with a Professor Jacobson, who had experimented
with the use of this substance in medicine. On the day of Han-
nover's departure, Jacobson had shown him a divided mammalian
eye, preserved in a solution of chromium trioxide. Hannover was
struck by the state of preservation of the organ, and on his return
home he tried hardening various tissues in 5% or even stronger
solutions and then cutting sections for microscopical study. He
found that the consistency was good for cutting. He mentions a
large number of tissue- constituents that were well preserved,
including blood-corpuscles, cartilage-cells, medullated nerve-
fibres, and ciliated epithelium from the mouth of the frog. His
paper ^^^ takes the form of a letter to Jacobson.

Chromium trioxide was used by Corti ^^^ in 1851 for the fixation
of the inner ear, and by Miiller ^^^ in i860, in a mixture with
potassium dichromate and sodium sulphate, to fix an abnormal
human eye. The discovery that it is useful in the fixation of chro-
mosomes was made by the Polish cytologist Mayzel ^^^ in 1878.
Strasburger *^^ in 1879 found a 1% solution the best fluid for
maintaining plant chromosomes in their living form, and he made
much use of it in his subsequent work. Flemming exploited
Mayzel's discovery from 1879 onwards. ^'^' ^^^ Much of his
pioneer work on chromosomes was done with chromium trioxide
at o-i to 0-2%.

Reactions with proteins. This is a powerful coagulant of
albumin, but it does not stabilize gelatine gels. It is exceptional
among fixatives in rendering proteins wholly resistant to digestion
by pepsin and trypsin. Chromium cannot be removed from tissues
even by prolonged washing, and it is probable that this is an
additive fixative; but very little is known about the chemical
changes that take place when solutions of chromium trioxide react
with proteins at room-temperature. Tyrosine, trytophane, and

PRIMARY fixatives: COAGULANTS IO7

histidine present side-groups that are capable of oxidation, and it
is noteworthy that after prolonged fixation by chromium trioxide,
histochemical tests for these amino-acids give feeble results. ^^^
This suggests that oxidation has in fact occurred, but it does not
explain the firm binding of chromium.

Chromium trioxide is a coagulant of nucleoprotein. DNA is
precipitated from its solutions in an insoluble form; ^^^ it is
hydrolysed, with conversion of the pentose sugar to aldehyde, and
Schiff's reagent (p. 308) therefore gives a positive reaction with-
out the necessity for hydrolysis by hydrochloric acid on the
slide.iii

Reactions with lipids. It was stated long ago by Smith and Mair ^^^
that the fat of adipose tissue (triglyceride) can be rendered in-
soluble in lipid-solvents by prolonged treatment with chromium
trioxide. This has recently been confirmed. ^^^ The reactions with
lipids have not been fully investigated. Unsaturated fatty acids are
first oxidized at the double bonds, with production of hydroxy-
acids.^2^ Oleic acid is eventually split, with production of pelar-
gonic and azelaic acids ; these are somewhat soluble in cold water.
Presumably the reactions with lipids in general are similar to
those of potassium dichromate (p. 128), but quicker and apt to go
too far. It may be for this reason that potassium dichromate is
nearly always used when tissues are 'postchromed' (p. 129) to
render lipids insoluble in lipid-solvents by partial oxidation.

Reactions with carbohydrates. When chromium trioxide acts on
polysaccharides, it works as an oxidizer, converting them to
aldehydes. The exact site of this reaction in the polysaccharide
molecule has not been determined. ^^^ The aldehvdes formed in
this way can be exhibited by means of SchiflP's aldehyde-reagent
(p. 308). This is the basis of Bauer's *^ histochemical test for
glycogen and other polysaccharides. It is usual to fix tissues in a
mixture of ethanol and acetic acid or some other fixative that will
not dissolve water-soluble polysaccharides, and then to treat
sections with a solution of chromium trioxide. If a simple aqueous
solution of chromium trioxide be used as fixative, a certain amount
of glycogen can subsequently be revealed in sections by the direct
application of Schiff's reagent. ^^^

If the proteins of a cell are fixed in such a way that the escape of
glycogen is hindered, and a solution of chromium trioxide is
subsequently allowed to act, the aldehyde produced will not dis-
solve out if the section be placed in distilled water. Nevertheless,

I08 FIXATION

it is doubtful whether chromium trioxide can be regarded un-
equivocally as a fixative for glycogen.

Polysaccharides that are not naturally chromotropic are
rendered so by the action of chromium trioxide. This subject will
be considered in the chapter devoted to metachromasy (see
especially p. 247).

Penetration. Chromium trioxide penetrates slowly into gelatine/
albumin gel (K = i-o), and slowly also into liver (K = 0-25).
Tellyesniczky's data give a higher K- value for liver, but he used a
1% solution. It will be seen from fig. 5, a (opposite p. 67) that
chromium trioxide penetrates liver more slowly than picric acid.

Shrinkage or swelling. Gelatine/albumin gels only shrink
slightly (to 91% of their original volume) in chromium trioxide
solution, and tissues in general are moderately shrunk. The
volume of the whole liver is reduced to 78% of the original and
there is further reduction to 64% in paraffin. The spermatocytes of
Helix, in paraffin sections mounted in Canada balsam, have 29% of
their original volume. In comparison with the other fixatives
tested by Ross, this is moderate shrinkage.

Hardening is moderate. Wetzel's figures show that acetone and
ethanol leave tissues about 20 times as hard as chromium tri-
oxide does, while the latter leaves them about 25 times as hard as
they are left by 10% acetic acid.

Immediate effects on particular constituents of the cell. As we have
see (p. 81), fixation by chromium trioxide is much improved by
the addition of an indifferent salt. It is unfortunate that in the
experiments of Strangeways and Canti and of Policard and his
colleagues, no such addition was made. Their results can be
briefly summarized thus. Pseudopodia, if present, are blunted ; the
ground cytoplasm is coagulated, sometimes rather coarsely; mito-
chondria are rendered invisible (and perhaps destroyed); lipid
droplets tend to fuse together. The nuclear membrane is rendered
clearly visible, the nuclear sap coarsely coagulated, the nucleolus
somewhat shrunken. Chromosomes are seen more clearly than in
life. The appearance of a cell before and after the addition of
chromium trioxide solution is shown in fig. 8, c, d (opposite
p. 70).

Methods of washing out. It is important to get rid of the excess of
the fixative, lest there should be reduction at some later stage to
green chromic oxide, CraOg, which is insoluble in ordinary solvents
and remarkably resistant to acids and other reagents. Since

PRIMARY fixatives: COAGULANTS IO9

ethanol might cause this reduction, tissues are usually washed in
running water before dehydration. Virchow ^^^ showed that no
insoluble precipitate was formed on direct transference of tissues
to 95% ethanol, if light was excluded. Overton ^"'^ recommended
the use of sulphurous acid after fixation. He thought that chromic
sulphate was formed: this, being stable and soluble, would be
harmless, and indeed would act as a mordant for certain dyes.
Direct transference from solutions of chromium trioxide to 50%
ethanol containing 2% of sulphuric acid is probably safe.^^

Effect on dyeing. Cytoplasm is rendered strongly acidophil by
chromium trioxide, while chromatin is fairly easily colourable by
most basic dyes. Differential dyeing of cytoplasm and chromatin is
therefore easy, though there is some tendency for acid dyes to
colour chromatin.

Effects on the general histological picture seen in parajfin sections.
The excellence of chromium trioxide for the fixation of the
neurones of Sepia, when dissolved at i % in sea-water, was noted
by Young. ^^^ In the presence of an indifferent salt at an appro-
priate concentration, this substance gives a better general picture
than any other primary fixative, with the possible exception of
chloroplatinic acid.^^ These two fixatives, with sodium chloride at
0*75%, fall into grade II. This grade is not reached by any
other primary fixative than these (though it could be reached
easily enough if paraffin embedding were not employed). In the
absence of sodium chloride, chromium trioxide falls into grade III.

The general picture given by chromium trioxide is as follows.
Cellular aggregates are fixed without serious distortion; cyto-
plasm is sometimes contracted round the nucleus, or coarsely
coagulated so as to leave wide meshes; mitochondria are not
fixed; red blood-corpuscles are swollen. The form of the nucleus
is well preserved; the nuclear membrane is clearly seen; the
nuclear sap is rather coarsely coagulated ; the nucleolus is well fixed
and has a strong affinity for both iron haematein and acid fuch-
sine; chromosomes are very well shown. The mitotic spindle
appears fibrous.

The concentration at which this fixative is used makes remark-
ably little difference. Even at o-oi% (with sodium chloride at
0*^5%)' chromium trioxide gives much the same picture as when
used at the ordinary concentrations. ^^^

Compatibility with other fixatives . It is best not to mix chromium
trioxide with substances that will reduce it, such as formaldehvde

no

FIXATION

or ethanol. The reaction with formaldehyde is very quick. The
reaction-products in the different mixtures containing chromium
trioxide and formaldehyde have not been identified. In Sanfelice's
fluid/^^ which contains these substances and acetic acid, the colour
of the reaction-products is brown; in Allen's *B.i5',^ which con-
tains picric acid and urea in addition, it is green. The latter colour,
combined with solubility, is suggestive of reduction to the ter-
positive ion in one of its hydrated forms.

Chromium trioxide is compatible with all the primary fix-
atives mentioned in this chapter and the next, other than
formaldehyde and ethanol, but when it is mixed with potassium
dichromate, the latter substance does not exhibit its own char-
acter (see p. 128).

Unclassified remarks. Bright light is destructive of cells fixed in
solutions of chromium trioxide. ^^ If the light of a dark-ground
condenser is focused on a cell while it still lies in the fixative, the
following changes may be observed. The outline of the cell be-
comes hazy; the particles of coagulated cytoplasm begin to show
Brownian movement and soon disintegrate, leaving only the lipid
globules and nucleus; the latter then dissolves, the nucleolus re-
sisting solution longer than the rest of it; finally nothing is left
except the lipid globules. *^^ The result is the same when heat- rays
from the microscope-lamp are filtered off.

The action of chromium trioxide as a fixative will be referred to
again in the section on potassium dichromate (p. 128).

CHAPTER 6

Vrimaiy Fixatives Considered
Separately 2. Non-coagulants

FORMALDEHYDE (methanal)
Standard concentration for fixation. 4% w/v aqueous solution.

"\ _

Formula and formula-weight. ^C — O. 30-0.

W

Description. Formaldehyde is a colourless gas. If liquefied it boils
at —19° C. It is very soluble in water and is commonly sold in
aqueous solution as 'formalin'. This is usually a 37% w/w solution
or thereabouts. The specific gravity of formalin is about i-o8, and
100 ml therefore contain almost exactly 40 g of formaldehyde.
Thus formalin is approximately a 40% w/v solution. Commercial
formalin contains a little formic acid (generally less than 0-05%)
and a considerable amount of methanol (6 to 15%).

Monomeric formaldehyde probably exists in water as
HOCHgOH. It has a strong tendency to polymerize as dimer,
trimer, etc., having the general formula H0(CH20)nH. In the
40% solution, only a small part (11%) of the formaldehyde is
monomeric, but when this is diluted to 4%, the monomer pre-
dominates. Paraformaldehyde tends to be deposited from concen-
trated solutions of formaldehyde as a white powder. This is a
highly polymeric form (w = 100 or more). The methanol in com-
mercial formalin hinders polymerization.

As Bethe '^^ pointed out 60 years ago, a considerable amount of
confusion is caused by the loose usage of the trade-name 'formalin'.
When authors say '10% formalin', they presumably mean form-
alin diluted with 9 times its volume of water or salt-solution to
give 4% w/v formaldehyde, but there is always the possibility that
they mean 10% formaldehyde. It is best to restrict the name forma-
lin to the commercial product at 40% and to express the concen-
tration of diluted fluids in terms of their formaldehyde content.

Ill

112 FIXATION

The only disadvantage in doing so is that the concentration of the
formaHn from which solutions are made up is not always exactly
40% w/v, and the concentration of formaldehyde in diluted solu-
tions is therefore not exactly known. Any error from this source,
however, is likely to be small.

The trade-term 'formol' is objectionable, for the ending in -ol is
unsuitable. 'Formal' is not allowable, because it is the accepted
name of another substance.

Ionization. Formaldehyde ionizes to a minute extent to form
hydronium ions,^^^ but acidity from this cause is negligible. The
acidity of formaldehyde solutions is due to oxidation to formic
acid by atmospheric oxygen. The pH of formalin is said to vary
from 3-1 to 4'i.^"^ The pH of the 4% w/v solution, made by dilut-
ing formalin with 9 times its volume of distilled water, is given by
various authors at figures varying from 3-4 to 4-6.112,397,176,459
The addition of 5 ml of pyridine to 100 ml of 10% formaldehyde
brings the pH to 7-0.^^ The 4% solution 'neutralized' by excess
of calcium carbonate has a pH of 6-4; ^^^ if basic magnesium car-
bonate is used the pH is 7-6.^^^ In the presence of 1% of calcium
chloride and excess of calcium carbonate the pH is 9-0; ^^^ this
is the most alkaline fixative used in microtechnique.

Oxidation-potential. Formaldehyde is capable of being reduced
to methanol according to the equation HCHO + 2H^ -\- 2e~ =
H3COH. It can thus act as an oxidizer, though a weak one. It was
claimed by Kingsbury -^^ long ago, in an important contribution
to the theory of fixation, that formaldehyde acts as an oxidizing
fixative. The oxidation-potential of the 4% solution is 0-23 volt.
This is the lowest of the oxidation-potentials of fixatives measured
by Casselman.ii^

Manufacture. Formaldehyde is made by passing a mixture of air
and vaporized methanol over a heated catalyst. Silver gauze at
635° C is suitable. Water formed by the reaction passes over and
dissolves the gas; also some unchanged methanol. The amounts of
these two substances are adjusted to give a solution at about
40% w/v, with as much methanol as is considered appropriate by
the manufacturer for protection against excessive polymerization.

Introduction as fixative. Formaldehyde was introduced into
microtechnique in 1893, later than any other important fixative.
The discovery of its fixative properties was made accidentally by
Blum, ^2 who had previously introduced it as an antiseptic. One day
he slit up a mouse that was infected with anthrax and left it over-

PRIMARY fixatives: NON-COAGULANTS II3

night in a solution of formaldehyde (presumably as a disinfectant).
In the morning he was surprised to find that it felt as hard as
though it had been preserved in ethanol. He then undertook a
systematic study of the effects on tissues, trying it at 4% on
various organs, including liver, kidney, the mucous membrane of
the stomach, and brain. He found that it hardened tissues faster
than ethanol and preserved their external form better, so far as
could be seen with the naked eye. He embedded various organs in
collodion, sectioned them, and found the cells well fixed and capable
of being dyed with haematein and synthetic dyes. It is perhaps
fortunate that he used collodion, because formaldehyde unmixed
with other fixatives gives rather poor results when tissues are em-
bedded in paraffin (p. 118).

Reactions with proteins. Formaldehyde does not coagulate
albumin; it renders this protein not coagulable by ethanol. ^^ Un-
like the coagulative fixatives described in the last chapter, it
stabilizes gelatine gels very perfectly, so that they do not dissolve
in water at 37° C but retain their form and transparency. There is
a slow gelatinizing action on histone.**^^ Haemoglobin is retained
in blood-corpuscles, perhaps with conversion to methaemo-
globin.*^^ Fixation by formaldehyde does not affect the action of
pepsin or trypsin on the proteins of blood plasma. There is
contradictory evidence about the ability of trypsin to digest
collagen fixed by formaldehyde.^^^' ^^^

The chemistry of the reactions with proteins has been
thoroughly investigated. Formaldehyde reacts with the -NHg of
the side-groups of certain amino-acids, probably forming methyl-
ene bridges that link protein chains together, lysine to lysine or
lysine to glutamine. The reaction is slow, especially below pH 3 :
the greatest binding of formaldehyde occurs at about pH 7-5 to 8.

Nucleoproteins are not coagulated; extremely minute flocculi
may appear eventually. DNA is not precipitated from its solu-
tions. ^^^ The amount of nucleic acids (DNA -|- RNA) in cells is
reduced by 10 to 35% by formaldehyde fixation.

Certain enzymes are not wholly inactivated by formaldehyde.
The enzymes in the liver of the rat have been studied in this
connexion. ^^^ /3-glucuronidase retained its activity best; then
sulphatase, acid phosphatase, and esterase ; alkaline phosphatase
least well.

Reactions with lipids. In general, formaldehyde is a good pre-
servative of lipids, including cholesterol and its esters, unless

H

114 FIXATION

fixation is very prolonged. Frozen sections are generally used, and
fixation of lipids (as opposed to their preservation) is therefore
unnecessary.

It has been known for many years that there is a gradual loss of
lipids if tissues are left in solutions of formaldehyde for a long
time.^^^' ^^^' ^^^' ^^ Cholesterol, triglycerides, and cerebrosides
appear to remain unaffected, but certain phospholipids begin to
disappear. Lecithins are well retained, but phosphatidyl serine,
phosphatidyl ethanolamine, and sphingomyelins become reduced
in amount. There appear to be two factors in the loss of phospho-
lipids. On one hand there is thought to be a splitting oflF of glycero-
phosphoric acid, the fatty acids remaining in the tissues; on the
other there may be a direct removal of lipid in colloidal solu-
tion. ^^^' ^^ In this connexion it is to be remarked that phosphatidyl
serine dissolves in 4% aqueous formaldehyde to form a clear
colloidal solution.^^

Phospholipids have a strong tendency to take up water and
extend their surface by growing outwards in worm-like 'myelin
forms'. This can easily be seen by smearing some lecithin across
the bottom of a cavity-slide and watching through the microscope
on the addition of water (fig. 15, a). The outgrowth of myelin
forms from the lipid constituents of cells would be damaging
morphologically and would also favour gradual solution. It was
shown by Leathes ^^^ that calcium ions have a remarkable effect in
preventing these outgrowths (fig. 15, b). The idea of adding cal-
cium chloride to formaldehyde solution therefore suggested it-
self. ^^ The salt has a double function: it checks the distortion and
solution of certain lipids, and it also improves fixation in the same
way as sodium chloride or other indifferent salts.

After treatment with formaldehyde, phospholipids are less
soluble in lipid solvents and therefore less extractable by these
from tissues.^^^'^^^'^^ Experiments on this subject may be carried
out as follows. ^^ Phospholipids are incorporated in elder-pith, and
the latter then placed in formaldehyde solution; frozen sections
are cut and treated with various lipid solvents, and then with one
of the colouring- agents for lipids (p. 299). In a series of experi-
ments of this sort, with formaldehyde at 4% in 1% (anhydrous)
calcium chloride solution as fixative, lecithins, 'cephalins', and
sphingomyelins were all rendered insoluble even in boiling ethanol
followed by boiling ether, and also in parafiin wax at 60° C pre-
ceded and followed by xylene. This shows that formaldehyde is

PRIMARY fixatives: NON- CO AGUL ANTS II5

a fixative for phospholipids, though not necessarily for mixtures of
these substances with other lipids. The chemistry of this process is
not known, but reaction between formaldehyde and the -NHg
group of phosphatidyl ethanolamine has been suggested. ^^^

If, after fixation with formaldehyde, less of a particular lipid is
extractable by lipid solvents than was present in the fresh organ,

a

d

1

1

•v"

\

■■

^

J

1

%'^/A'

;^4

V,"

><4-

Irr^

r

B

FIG. 15. Photomicrographs of lecithin smeared on glass.

A, in distilled water, showing the outgrowth of myelin forms. B, in a concentrated solu-
tion of calcium chloride, showing absence of myelin forms.

In each case the photomicrographs were taken a, before the addition of fluid; b—f after
the addition, at intervals of 2 j, 9, 17, 33, and 57 minutes respectively.

(From Leathes,^'" by kind permission of the Editors and Publishers of The Lancet )

two very different explanations are possible. On one hand the
lipid may have partly dissolved out into the formaldehyde solu-
tion; on the other it may have been rendered insoluble in lipid
solvents and therefore not accessible to ordinary methods of
analysis.

It is claimed by Wolman and Greco ^^^ that formaldehyde
reacts with unsaturated lipids at the double bond (-C==C-),
producing carbonyl groups that w^ill react with Schiff's aldehyde-
reagent (p. 308). The capacity of the lipid to take up iodine is at
the same time reduced, and this suggests that the double bond has
been attacked by formaldehyde. If reagents that block carbonyl
groups are used, there is no longer a positive result with Schiff's
test.

Il6 FIXATION

Glycerides and fatty acids are sometimes crystallized and thus
rendered anisotropic by the action of formaldehyde. Cholesteryl
esters are transformed from liquid spherocrystals, showing the
cross of polarization, into simply anisotropic, solid crystals.^^*^

The colouring agents for lipids (p. 299) are said to act particu-
larly strongly when formaldehyde is used as fixative.''^

Reactions with carbohydrates. Formaldehyde does not fix soluble
carbohydrates, but it has a remarkable capacity to fix proteins in
such a way that the escape of glycogen by solution in water is
hindered. ^^^ It may be remembered that picric acid has a similar
property (p. 97).

Rate of penetration. Formaldehyde enters gelatine/albumin gel
faster than any other fixative except the strong mineral acids, at a
K- value of 3-6. The penetration of non-coagulant fixatives into
tissues is hard to measure. Tellyesniczky's data give a K-value of
0-78. This is only a moderate speed, equal to that of mercuric
chloride.

Shrinkage or swelling. Gelatine/albumin gels swell consider-
ably (to 123% of their original volume) in formaldehyde solution.
The volume of whole liver remains almost unchanged (99%), but
there is subsequent shrinkage to 68% by the time the organ is in
paraffin.

Single cells may be observed to shrink at first contact with
formaldehyde solutions, especially if the latter are rather con-
centrated; this is usually followed by expansion at some time
during the first hour to a considerably greater volume than the
original, and later by a second shrinkage that leaves the cell
somewhat larger than it was in life.^^* The expansion after the
original shrinkage is less marked if the formaldehyde is used in
saline solution, and the cell may eventually return to its original
size. The pulsation of nuclei is less than that of the cytoplasm, and
not exactly synchronized with it.

Most observers have not noticed this curious pulsation, but have
simply recorded the final size when fixation is complete. Arhacia
eggs swell strongly (to 147%) in formaldehyde dissolved in dis-
tilled water. They swell slightly (to 105%) when it is dissolved in
sea- water, but the volume is down to 48% in xylene. Helix sperma-
tocytes have a volume of 34% of the original in paraffin sections
mounted in Canada balsam. It is a sobering thought that this
represents less shrinkage than that obtained with any other pri-
mary fixative.

I

PRIMARY fixatives: NON-COAGULANTS II7

Harde?ting. Formaldehyde hardens strongly. Wetzel, using a 10%
solution, obtained a rigidity-figure of about 1700. This is 7 J times
the figure for chromium trioxide and is exceeded only by ethanol
and acetone.

Immediate effects on particular constituents of the cell. The cell
outhne is often well preserved, but there is a strange tendency for
blebs of cytoplasm to separate from the cell. The ground cyto-
plasm is not so homogeneously fixed as experiments with gelatine
and gelatine/albumin gels would lead one to expect: there is a
tendency for a certain amount of granulation to occur. Mito-
chondria are preserved, often rather well, though sometimes they
become moniliform. When once they have been fixed by form-
aldehyde, they are no longer subject to destruction by acetic
acid.^^^ Lipid globules usually remain as in life, but a wide variety
of these has not been studied.

The shape and structure of the nucleus are on the whole well
preserved, though there is some tendency for the nuclear sap to
become granular. The heterochromatic segments of the chromo-
somes remain more or less as in life. The nucleolus is less clearly
seen than before fixation.

There can scarcely be any doubt that formaldehyde (with an
indifferent salt) preserves the structure of the living cell better
than any other primary fixative except osmium tetroxide.

Methods ofzvashing out. As a general rule, no special washing out
is necessary. Tissues may be transferred to water or to ethanol of
any grade.

Dark brown, birefringent crystals are sometimes seen in tissues
that are rich in blood, especially spleen. This so-called Tormalin-
pigment' appears to arise by the reaction of formaldehyde with
the haematin of haemoglobin that has escaped from red blood-
corpuscles either before or after death. It is not formed if short
fixation in formaldehyde solution is followed by prolonged soaking
in 5% mercuric chloride solution. ^^^ Once formed, it can be dis-
solved by a 1% solution of potassium hydroxide in 80% ethanol,
or by picric acid dissolved in ethanol. ^-^ The chemistry of these
processes has not been worked out.

Effect 071 dyeing. Formaldehyde renders proteins and cytoplasm
more acidic (basiphil) than any other fixative, exceeding mercuric
chloride in this respect: cytoplasm retains little affinity for acid
dyes unless fixation is short. Chromatin is strongly coloured by
basic dyes.

ii8

FIXATION

Effects on the histological picture seen in paraffin sections. Form-
aldehyde is a poor fixative for tissues that are to be embedded in
paraffin (grade IV-V if dissolved in distilled water, III-IV if
sodium chloride is added at 0-7%).

Cellular aggregates tend to be widely separated from one
another; cytoplasm shrinks towards nuclei, and cells may lose con-
tact with one another; mitochondria are sometimes retained,
especially if the fixative be used in strong solution (10% or more).*^
Red blood-corpuscles tend to swell into spheres, but are rather
well preserved if an indifferent salt is mixed with the fixative. The
interphase nucleus is fixed in a remarkably life-like form, with no
coarse network in it; ^^^ the nuclear membrane, heterochromatic
segments of the chromosomes, and nucleolus are well shown. The
mitotic and meiotic chromosomes, however, are very poorly fixed.

Formaldehyde gives good results if frozen sections are used, or
tissues embedded in collodion.

Compatibility with other fixatives. Formaldehyde reduces chro-
mium trioxide quickly, potassium dichromate much more slowly.
The reaction with osmium tetroxide is very slow at room-tem-
perature. ^^ Formaldehyde is compatible with ethanol, picric acid,
and acetic acid, and is generally regarded as compatible with
mercuric chloride.

OSMIUM TETROXIDE

Standard concentration for fixation. 1% w/v aqueous solution.

Formula and formida-weight. OSO4. 254-2.

Description. Osmium tetroxide occurs as pale yellow crystals.
These melt at 41° C. The liquid boils at 131° C, but much vapour
comes off before this temperature is reached. The vapour arises
also from the crystals and from aqueous solutions ; it is damaging
to the epithelium of eyes, nose, and mouth. This vapour has a not
unpleasant smell. The name of the metal is derived from the fact
that the tetroxide smells. The volatility of osmium tetroxide makes
it imperative to keep and use solutions in tightly-stoppered
vessels. ^^^

The solubility in water at 25° C is 7-24% w/w.^^ (The figure
given by Thorpe and Whiteley,^^^ who are usually so reliable, is
grossly in error.) Osmium tetroxide is extremely soluble in carbon
tetrachloride at 25° C (about 375% w/w ^^) and soluble also in
liquid paraffin and certain lipids.

PRIMARY fixatives: non- CO agulants 119

Most compounds of osmium are dark or black. On reduction,
osmium tetroxide is converted to the grey or black anhydrous
dioxide, OsOg, or to the brown, gelatinous, hydrated dioxide,
OSO2.2H2O or Os(OH)4.^^^ Reduction takes place readily when
solutions of osmium tetroxide are exposed to light, though it is
claimed that this is prevented by the complete exclusion of dust.^^
Reduction by light is prevented by strong oxidizers (p. 125), and
is also hindered, curiously enough, by sodium chloride. ^^

Ionization. Osmium tetroxide scarcely ionizes. It was recognized
long ago that the electrical conductivity of its solutions was ex-
tremely low. Hofmann and his colleagues ^^^ found that the
specific conductivity of a 1% solution in distilled water was
1-09 X 10"^; that of the water used was 0-5 X io~^. 'Osmium
tetroxide', thev remarked, 'is therefore chemicallv neutral and no
acid.'

On solution, osmium tetroxide takes up a molecule of water
and becomes HoOsOj.^*^ This substance cannot be isolated.
A minute amount of ionization occurs, according to the
formula HoOsOj-*-^- H^ + HOsOj". The ionization constant is
8-0 X io~^^. ^^^ It follows from this that 100 ml of a i % solution of
osmium tetroxide contain 0-000000035 § ^^ hydrogen ion. To call
such a substance an acid seems rather far-fetched, though a
sodium salt, NaHOsOj, can in fact be obtained. Hydrogen
peroxide has an ionization constant of 1-78 X lO"^-, **^ more than
tw^ice that of HoOsOs, but it is not called hydroperoxidic acid.
The pH of a solution of osmium tetroxide is almost exactly that of
the distilled water used to make it. The values published in the
biological literature give us information about the distilling
apparatus or glassware used in various laboratories, but not about
osmium tetroxide.

The proper name for HoOsOs is not easy to determine. It cannot
be osmic acid, for on the analogy of manganic acid (HgMnOJ,
osmic acid is H2OSO4; and this is a real entity, as we have seen
(p. 62), though it is not formed when osmium tetroxide is dis-
solved in water. Permanganic acid is HMn04, and HgMnOs
would be per-permanganic acid. On this analogy HgOsOs would
be per-perosmic acid,^"*^ if it were unequivocally an acid. It seems
best to call it hydrogen per-perosmate.

Oxidation-potential. The 2% solution has an oxidation-potential
of 0-64 volt, somewhat less than that of a 5% solution of mercuric
chloride.

120 FIXATION

Manufacture. Metallic osmium occurs naturally as an alloy with
iridium, usually in association with platinum. Deposits are found in
Alaska, the Ural Mountains, and in South Africa. There is an
ounce of osmium in 1,200 tons of Witwatersrand gold ore. The
metal is extremely heavy (density about 22-5). It is also very hard
and its alloys are used in making pivots for scientific instruments
and tips for nibs of fountain pens.

Osmium tetroxide is made by heating spongy metallic osmium
in a current of air or oxygen. On account of the rarity of the metal,
this fixative is extremely expensive. A gram costs ^3. 10. o; thus
I ml of a 2% solution costs 1/4 J, and a single drop of it about f^.

Introduction as fixative. We are indebted to Franz Schulze of
Rostock, the inventor of that invaluable histochemical reagent,
chlor-zinc-iodide,**^ for the introduction of osmium tetroxide into
microtechnique. Unfortunately it is impossible to discover what
led him to try it. He noticed that different tissue-constituents
differed in their capacity to reduce it to the dark lower oxide. He
sent a weak solution (o-i or 0-2%) to his friend and former pupil,
Max Schultze, with the request that he should try it in histological
investigations. Schultze did so in 1864.^** He plunged the male of
the beetle Lampyris splendidula, alive and shining, into Schulze's
fluid, and made a microscopical study of the phosphorescent organ.
To his surprise, the tracheal end-cells were blackened and thus
showed up strongly against the parenchymal cells.***' **^ In
collaboration with Rudneff **^ he next tried it on a variety of
tissues of plants and animals. He noted especially the reduction of
osmium tetroxide by fat, myelin, and tannic acid. It is interesting
that his emphasis was at first on the darkening of particular
objects, not upon delicacy of fixation. Retaining his interest in
phosphorescence, however, he tried it on the marine protozoon
Noctiluca, and was now struck by the life-like preservation.**^

Reaction with proteins. Osmium tetroxide gives no coagulum
with albumin solutions. It renders albumin not coagulable by
ethanol or by heat.^^ It sets undiluted egg-white and strong solu-
tions of serum albumin, serum globulin, and fibrinogen into gels.
It stabilizes gelatine gels against solution by water at 37° C.

This is an additive fixative. It probably reacts at the double
bonds of the side-groups of tryptophane and histidine, linking
protein chains together through these. The failure of acid dyes to
act after fixation by osmium tetroxide suggests the blocking of
amino-groups, but there is no positive evidence of this.

PRIMARY fixatives: NON - CO AGUL ANTS 121

There is no coagulation of nucleoprotein, nor is DNA pre-
cipitated from solution. ^^^

Reactions with lipids. As we have seen (p. 120), Schultze and
RudnefT *'*^ had already in 1865 recognized the capacity of fats
and other lipids to reduce osmium tetroxide. It was shown by
Altmann ^ that whereas oleic acid and olein are blackened by this
substance, palmitic and stearic acids and their triglycerides are
not. This led to the understanding that osmium tetroxide reacts
with the double bonds in lipids. Since lipids generally occur in
nature as mixtures, and some of the constituents of these mixtures
are usually to some degree unsaturated, most lipids as they occur
in organisms will sooner or later be darkened by the action of
osmium tetroxide. As a general rule, mixed triglycerides in the
form of storage-fat blacken with osmium tetroxide more easily than
conjugated lipids.

When osmium tetroxide reacts with lipids, three possibilities
present themselves. The tetroxide may be reduced to the dark
lower oxide ; or it may combine with the lipid to form a dark com-
pound; or both may occur. If a compound is formed, alteration
in solubility is to be expected. It is well known that the sites of
storage fat are often black in paraffin sections mounted in Canada
balsam. Lipids are unlikely to survive such embedding and
mounting, unless profoundly altered. The possibility presents
itself, however, that the lipid has gone and only insoluble osmium
dioxide remains to mark its former sites.

Comments on this subject are scattered through the literature.
Recently the Chinese cytologist Chou,^^^ working at Oxford, has
made a systematic study of the subject. The subcutaneous fat
of the mouse was used in his experiments. This was fixed in
1% osmium tetroxide or in Flemming's strong fluid, ^"^ which
contains osmium tetroxide at 0-4%. The fixed tissue was de-
hydrated with ethanol, left for 30 min. in an antemedium, and
embedded in paraffin. Sections were soaked for 5 min. in the same
fluid that had been used as antemedium, brought down to water,
and mounted in an aqueous medium. The antemedia used were
xylene, toluene, benzene, and chloroform.

The fat-oites were in all cases black. If, however, the sections
were left in xylene or toluene for 40 instead of 5 min., the fat-sites
were colourless: the globules appeared empty. They remained
black, however, if benzene or chloroform was used for the same
period.

122 FIXATION

When sections that had been 5 min. in any of the antemedia
were brought down to water and bleached, it could easily be
shown that the lipid still remained in them, for it could be coloured
black by Sudan black. If, however, sections were left for 40 min.
in the antemedia, the ones treated with xylene or toluene were
proved to contain no lipid, while those that had been in benzene or
chloroform still contained it.

Sections that had been 5 min. in the antemedia were brought
down to water, bleached with hydrogen peroxide, taken up to the
antemedia again, and left there for 30 min. On subsequent treat-
ment in the usual way with Sudan black, they were shown to
contain no lipid. This applied in all cases, whether the antemedium
was xylene, toluene, benzene, or chloroform.

It is to be noted that so long as the lipid-sites were still blackened
by osmium, they contained lipid. The evidence suggests strongly
that the black substance is a compound of lipid with osmium.
This compound is resistant to solution by benzene and chloroform,
but dissolves slowly in xylene or toluene; when bleached by
hydrogen peroxide, it is soluble in any of the lipid solvents.
Xylene is capable of acting as an oxidizing agent,^-^ and the same
may perhaps apply to toluene. It is to be supposed that osmium
tetroxide reacts with the double bonds of lipids in much the same
way as it does with those of the substances discussed on pp. 61-2,
but no direct evidence on this subject is available.

The possibility that osmium tetroxide sometimes simply
oxidizes lipids, without forming an additive compound, must be
kept in mind. Hofmann ^^^ believed that this was the way in which
it ordinarily reacted with lipids. He made a careful study of oxida-
tion by osmium tetroxide, and reached the conclusion that it can
act as an adjuvant to other oxidizers. Thus, certain substances
that cannot be oxidized by potassium chlorate alone, can be
oxidized if osmium tetroxide is present as well. The latter oxidizes
the substrate and is itself reduced: the chlorate re-oxidizes it, and
the process begins again. Wolman ^*^ suggests that osmium tetrox-
ide may act as an oxidative catalyst in this way in microtechnique,
when mixed with other oxidizers.

In the presence of a strong oxidizer, such as chromium trioxide,
the mixed triglycerides of adipose tissue are generally blackened by
osmium tetroxide, while conjugated lipids as a rule are not. This
is useful as a rough pointer in preliminary histochemical work.
Since the fatty acid component of conjugated lipids is often highly

I

PRIMARY fixatives: NON - CO AGUL ANTS I23

unsaturated, it is not clear why they remain colourless. Perhaps
they tend to be simply oxidized instead of forming additive com-
pounds.

Since osmium tetroxide is soluble in certain lipids, it can be
taken up without change by fully saturated ones, and then reduced
by subsequent soaking of the tissue in ethanol (compare Starke ^^^).
Thus the fact that a lipid is black in a paraffin section does not
prove that it is unsaturated.

Reactions with carbohydrates. It appears that osmium tetroxide
does not react with most hexoses or pentoses or their polymers at
room-temperature, though sucrose is very slowly oxidized to
oxalic acid.^^ There is some darkening if glycogen is treated with
osmium tetroxide for long periods at 50° C.^^

Rate of penetration. Osmium tetroxide penetrates slowly into
gelatine/albumin gel. The K-value for 25 hours is 0-85, which is
nearly as low as the figure for picric acid. The K-value gradually
falls off with time. During the first 16 hours it is i-o, but during
the period 16 to 144 hours it is only 0-31. It must be supposed that
the osmium deposited in the gel (whether in combination with
protein or in the form of the dioxide) presents an obstacle to
diffusion. A measurable fall-off in K-value is not known to occur
with any other fixative.

Tellyesniczky's data ^^^ for penetration into liver (12 hours) give
K- values of 0-29 for the 0-5% solution and 0-58 for the 2%. This
indicates that the i % solution would be one of the more slowly
penetrating fixatives.

Shrinkage or swelling. Gelatine/albumin gel shrinks very slightly
(by less than 10% of its volume) in osmium tetroxide solution.
The change of volume of whole livers has not been measured,
presumably because the experiment would be too expensive. It is
unfortunate that there are no satisfactory numerical data for the
shrinkage or swelling of cells. Kaiserling and Germer ^^^ found
that mammalian eggs increased somewhat in diameter when trans-
ferred from saline to osmium tetroxide dissolved in distilled water.
The saline was hypotonic and the eggs had already swollen some-
what in it.

Hardening. Osmium tetroxide leaves tissues rather soft. Wetzel's
figure is 171 ; the figure for chromium trioxide is 1-4 times greater.
Tissues fixed in osmium tetroxide are crumbly in paraffin and do
not section well.

Immediate effects on particular constituents of the cell. By common

124 FIXATION

consent of all who have studied the subject, osmium tetroxide
preserves the structure of the living cell better than any other
primary or mixed fixative. Fig. 8, A, B (opposite p. 70) gives a
good impression of its action. The nuclear sap and the ground
cytoplasm in the vicinity of the nucleus become less perfectly
homogeneous than they were in life; the nucleus may retract
slightly from the cytoplasm; nucleoli become difficult to see;
lipid globules gradually darken. Until the latter change has taken
place, one might almost suppose that the cell was still alive, except
that any Brownian movement will have ceased. Mitochondria are
perfectly preserved.

Methods of washing out. Osmium tetroxide is washed out in
running water, because if any were left in the tissues, it might be
gradually reduced by ethanol at subsequent stages, with con-
sequent darkening.

Effects on dyeing. Osmium tetroxide leaves cytoplasm readily
colourable by basic dyes (after bleaching), but scarcely at all by
acid ones. The nuclear sap also tends to be made basiphil, and this
interferes with the differential dyeing of the component parts of
the nucleus.

Effects on the histological picture seen in paraffin sections. It is sad
to turn from the magnificent view of a cell still lying in osmium
tetroxide solution, to look at a paraffin section of a piece of tissue
fixed in the same ffuid. The fixation is poor (grade III-IV or IV),
even with the addition of 0-7% of sodium chloride to the fixative.

Cellular aggregates are severely shrunken, so that they are
separated by wide artificial spaces; cracks often run at random
across the section ; ground cytoplasm, though fairly homogeneous,
is strongly contracted, and often condensed round nuclei. The
shape of nuclei is w^ell retained. Pischinger ^^^ considered that the
nucleus as a w^hole was well fixed. He thought that there was no
nuclear membrane in the living cell but only a physical interface,
and that while other fixatives thickened the interfacial region to
form an artificial nuclear membrane, osmium tetroxide provided
an approximation to the living condition. The nuclear sap is rather
homogeneously fixed, but the objects contained in it (especially
the meiotic chromosomes) are very poorly shown.

Mammalian testis fixed in osmium tetroxide solution buffered
at pH 7*4 is shown in fig. 9, b (opposite p. 74).

Compatibility with other fixatives. Osmium tetroxide is com-
patible with all the fixatives mentioned in this chapter and the

PRIMARY fixatives: NON-COAGULANTS 125

preceding one, except formaldehyde and ethanol. The reaction
with formaldehyde is slow: no darkening occurred within 24 hours
at 20° C in the circumstances of Bahr's experiments.^^ Mercuric
chloride, chromium trioxide, and potassium dichromate prevent
the reduction of osmium tetroxide by daylight.

Unclassified remarks. It w^as first pointed out by Altmann * in
1889 that lipid globules are sometimes only blackened by osmium
tetroxide on the outside, so that they appear as rings in optical
section. He called these Ringkorner. He found that they were not
seen initially, but only when tissues fixed in osmium tetroxide had
been brought into ethanol. He attributed their formation to partial
solution of the lipid droplet by ethanol.^

Mmm^..,-----I(apuze (rettkornrest)
4^^g. Lucke ■ ■ 'Wmm- Proioplasmo.

FIG. 16. Three Ringkorner and a cap or hood (Kapiize) formed by partial
solution of lipid globules: osmium preparations.

(From Starke.-'^')

This subject was carefully investigated by Starke. ^^^ He found
that when lipid droplets that had been treated with osmium tetrox-
ide were set free in ethanol, they never became Ringkorner, but
shrank into irregular shapes, blackened all through. When similar
droplets were treated with osmium tetroxide while still contained
in the tissues, the result was different; for when the tissues were
subsequently placed in ethanol, Ringkorner were formed. Starke
concluded that lipid droplets consisted of a part that was rendered
insoluble in ethanol by osmium tetroxide, and a part that was not.
When the latter was dissolved out by ethanol, the droplet shrank if
it could ; but if it were surrounded by fixed cytoplasm it could not
shrink, and a spherical hole was left, to the walls of which the
fixed and blackened lipid material attached itself (fig. 16).

The black rings and crescents commonly seen in osmium pre-
parations are in many cases to be attributed to the cause sug-
gested by Starke. His paper, published more than 60 years ago, has
unfortunately been overlooked by many authors.

Pieces of tissue that have already been fixed in another fixative
(generally a mixture containing osmium tetroxide) may be soaked
for several days in a simple aqueous solution of osmium tetroxide.

126 FIXATION

to darken certain cytoplasmic inclusions. This process of 'postos-
mication' was introduced by the Polish cytologist Weigl.^^^ It is
useful for directing attention to a particular part of a cell, but it
should be used with caution. Ringkorner are often seen in post-
osmicated preparations. There is a tendency for a black material
(presumably osmium dioxide) to be deposited on the surfaces of
granules or other cytoplasmic inclusions, and especially to fill up
the spaces between crowded granules. The appearances given can
be very misleading morphologically, and should not be trusted
unless they can be confirmed by study of the living cell.

POTASSIUM BICHROMATE

Standard concentration for fixation. 1-5% w/v aqueous solution.

Formula and formula- weight. K2Cr207. 294-2.

Description. Potassium dichromate crystallizes readily in large,
orange-red prisms or tables. These melt with decomposition at
396° C. They are soluble at about 10% w/w in water at room-
temperature (18% at 30° C), but insoluble in absolute ethanol.

Potassium dichromate is more expensive than the sodium salt,
but the fact that it is anhydrous and not deliquescent gives it an
advantage for certain industrial purposes. It is used in making
matches and fireworks and in the chrome tanning of leather ; dis-
solved with sulphuric acid it acts as a bleaching agent for tallow
and palm oil.

It is wrong to call this substance potassium bichromate, because
the name would only be applicable to potassium hydrogen chro-
mate, which does not exist.

Ionization. The ionization of potassium dichromate has been
carefully considered by Casselman.^^^ The ions are the same as
those produced by chromium trioxide, but in different proportions.
The ions in solutions of the two substances containing the same
weight of chromium are compared in fig. 14 (p. 105). In both
solutions by far the greater part of the chromium is in the form of
dichromate [Cr207]" and hydrogen chromate [HCr04]~, the
former predominating in both cases, especially in the solution of
potassium dichromate. The chromate ion [CrOJ" is present in
minute quantities in both. Undissociated chromic acid, H2Cr04,
is present in considerable amount in the solution of chromium
trioxide, but there is scarcely any of it in the solution of potassium
dichromate.

PRIMARY fixatives: NON-COAGULANTS 127

There is a striking difference between the hydronium-ion
concentration of the two solutions. A 2-5% solution of potassium
dichromate has a pH of 4-05 ; in a solution of chromium trioxide
containing the same amount of chromium as the dichromate solu-
tion the pH is 0-85.^1^

If a solution of potassium dichromate be acidified to the same
pH as a solution of chromium trioxide containing the same
weight of chromium, the ions present in the two solutions will be
the same, except that the former will contain potassium ions and
the anions of the added acid. If hydrochloric acid be used, one has
a fluid almost identical with a solution of chromium trioxide to
which some potassium chloride has been added. Since potassium
and chloride ions are inactive in fixation, it follows that an acidified
potassium dichromate solution will act like a solution of chromium
trioxide.

Casselman does not give the pH of a 1*5% solution of potassium
dichromate, but a 1% solution (pH 4-10) difi'ers only slightly from
the 2-5% solution. Lassek ^^^ gives pH 4-0 for Miiller's fluid,
which is 2 or 2-5% potassium dichromate with 1% sodium sul-
phate (see below).

Oxidation-potential. The oxidation-potential of a 3% solution
of potassium dichromate is 0-79 volt.^^^ A 2-5% solution has
almost exactly the same oxidation-potential.^^^

Manufacture. Chromium occurs naturally as chromite,
FeO.Cr^O.^. It is treated with sodium carbonate to produce
sodium chromate, and this with sulphuric acid to give sodium
dichromate. Potassium chloride is added to a strong, warm
solution of the latter, and large crystals of potassium dichromate
separate out on slow cooling, or small ones if the tank is
shaken.

Introduction as fixative. Potassium dichromate was introduced
into microtechnique in i860 by H. Miiller,^^^ who used it in
studies of the human eye. His first fluid consisted of this salt and
sodium sulphate (presumably Glauber's salt, crystallized with 10
molecules of water), both at about 1*5%, with etwas chromium
trioxide. Later in the same year ^^^ he mentioned another fluid,
from which the chromium trioxide was omitted ; the concentration
of the other components was not stated, but was presumably the
same as before. The fluid called Miiller'sche AugenflUssigkeit con-
sists of the same two salts, at 2-2^^% and 1% respectively.^ ^^ It
forms the basis of Zenker's ^^* and Helly's ^^* fluids.

128 FIXATION

The fact that potassium dichromate is unsuitable for use in
studies of mitosis was first made known by Mayzel.^^^

Reactions with proteins. This is a non-coagulant of albumin
solution, but it very gradually renders undiluted egg-white more
viscous and eventually transforms it into a weak, semi-transparent
gel.

Since, as we have seen (p. 126), the chrome anions are almost
the same whether potassium dichromate or chromium trioxide be
dissolved, it must be supposed that the striking differences be-
tween the effects of the two substances on proteins must be due to
the large difference in pH. If potassium dichromate be acidified, it
reacts with proteins like chromium trioxide: that is to say, it
becomes a strongly coagulant fixative. The change-over from one
behaviour to the other occurs in the pH-range 3-4 to 3*8.^^^ As
Casselman points out,^^^ this is near to the iso-electric points of
many proteins (though somewhat below that of most). It must be
supposed that in the region of the iso-electric point, the proteins
change radically in their reactions to the chrome anions. The
chemical changes concerned in the slow gel-forming process that
occurs above the critical pH-range have not been investigated.

If gelatine that has been impregnated with potassium dichromate
is exposed to light, it becomes insoluble in warm water. This fact is
used in the *Autotype' process of photographic printing. It is
interesting to notice that chromium trioxide, on the contrary,
makes protoplasm soluble on exposure to bright light (p. no). In
the ordinary circumstances of fixation, gelatine/albumin gel is not
stabilized against warm water by the action of potassium
dichromate.

Nucleoprotein solution is not coagulated by potassium dichro-
mate, and DNA is readily dissolved by this salt; the histone of the
nucleus, however, is strongly gelatinized by potassium dichro-
mate. ^^^ There is a marked contrast here with the effect of acetic
acid, which precipitates DNA but dissolves histone (see p. 135).

Reactions zvith lipids. Potassium dichromate is able to attach
chromium to certain lipids, and to render them insoluble in lipid
solvents. The chromium can subsequently be made to react with
haematein to give a black lake. This is the essence of Weigert's ^^^
method for myelin. Tissues were fixed in Miiller's fluid, embedded
in collodion, and treated with a solution of haematein (often called
Weigert's 'haematoxylin', but haematoxylin is not a dye (see
p. 173)). Myelin was coloured black. Other tissue-constituents

PRIMARY fixatives: NON- COAGULANTS 129

were darkened by unmordanted haematein; this was removed by
bleaching with alkaline potassium ferricyanide, which did not
affect the lake.

Better results were obtained by short fixation in some other
fluid and subsequent 'postchroming' in potassium dichromate
solution. It is usual to postchrome tissues for quite a long time,
often days or weeks, sometimes at 37° or even 60° C. This process,
as a method of fixing particular constituents of cells, was intro-
duced by the celebrated German cytologist Benda,^^ who used it in
his pioneer research on mitochondria. He named it PostcJiro?ninmg.
It had previously been used only to harden tissues for easier
sectioning by hand. Benda sometimes used chromium trioxide in
the same way, but potassium dichromate is nearly always used
now^adays.

Smith *^^' *^^ modified Weigert's method by introducing a pre-
liminary fixation in formaldehyde, followed by postchroming and
the cutting of frozen sections. This technique was adapted by
Dietrich ^"^^ and made into a histochemical test for phospholipids.
The acid haematein test ^^'^^ is its modern version.

The chemistry of the action of potassium dichromate on lipids
has been studied especially by Kaufmann and Lehmann 260-262
and by Lison.^^^ It would appear that a wide variety of unsaturated
lipids can be rendered insoluble in lipid solvents by the prolonged
action of potassium dichromate. There is no action on saturated
ones. Different periods of postchroming are suitable for different
unsaturated lipids.

The evidence suggests that three processes can be involved in
the action of potassium dichromate on lipids, and that they need

H H H H

— C=C— ► — C— C— ► — CH HC—

\ \ II II

0—0 o o

Double bond Peroxide Aldehydes

not all occur together. These three are simple oxidation, poly-
merization with loss of solubility in lipid solvents, and binding of
chromium (additive fixation).

Simple oxidation at the double bond occurs particularly when
there is only one such double bond in a fatty acid radicle. The
fatty acid chain is split at the double bond, with formation of two
aldehydes.

130 FIXATION

Polymerization is particularly apt to occur when a double bond
lies near the opposite end of a fatty acid chain to the carboxyl
group. Oxidation proceeds as far as the peroxide stage, and mole-
cules then associate to form a polymeric, insoluble substance.

When a fatty acid radicle is highly unsaturated, the double bond
nearest to the carboxyl group behaves in a special way. Oxidation
proceeds as before to the peroxide stage; there is then a passage

0—0 00 o ox

Peroxide Diket07te Chromium compound

through dihydroxyketone to diketone, and chromium is then
taken up as oxide. This oxide, of unstated composition, is show^n as
X in the formula given here.

In Lison's view,^^^ phospholipids take up chromium because
their fatty acid components tend to be particularly highly un-
saturated.

One of the most valuable properties of potassium dichromate is
its ability to fix mitochondria by rendering their lipid components
insoluble in lipid solvents.

It is important to bear in mind that triolein, according to Smith
and Thorpe, ^^^ can take up chromium if postchroming is pro-
longed, and become insoluble in alcohol, xylene, and ether; even
storage-fat can be made to give a black reaction with haematein.*^^
Kaufmann and Lehmann '^^"^ claimed that all unsaturated lipids
could be rendered insoluble in lipid solvents by potassium di-
chromate. Chou ^^^ has recently obtained some confirmatory
results. He has shown that if the subcutaneous adipose tissue of
the mouse be fixed in Ciaccio's fluid ^^^ and then left in a saturated
solution of potassium dichromate at 37° C for 49 days, the fat is
rendered insoluble in certain lipid solvents. The tissue can be
dehydrated and brought through xylene into paraffin: the fat
globules can be deeply coloured with Sudan black in sections of
such material. Nevertheless, potassium dichromate does not
compare with osmium tetroxide in ability to render most lipids
insoluble, and storage fat is not ordinarily preserved in paraffin
sections of tissues fixed with potassium dichromate.

It seems probable that lipids are more widely dispersed in the
ground cytoplasm than is usually supposed, and potassium
dichromate may fix partly by acting on these.

PRIMARY fixatives: NON-COAGULANTS 13I

Reactions with carbohydrates. Potassium dichromate is not a
fixative for glycogen. Chromium is not known to be taken up from
solutions of potassium dichromate by any carbohydrate or related
substance, with the possible exception of lignin.^^ It is to be pre-
sumed, however, that acidified potassium dichromate will react
towards carbohydrates in the same way as chromium trioxide
(p. 107).

Penetration. Telly esniczky's data give a high K- value (i'33) for
the penetration of the 3% solution into liver, but it is doubtful
whether this means much. The term rate of penetration, as used in
this book, means the rate of penetration with fixative effect. Now
potassium dichromate does not coagulate proteins, nor does it
gelatinize most of them in the ordinary period of fixation. Figures
for rate of penetration do not seem to be applicable to this sub-
stance, though no doubt it runs quickly through protein gels ^^^
and tissues.

Shrinkage or swelling. Gelatine/albumin gels swell strongly (to
160% of their original volume) in potassium dichromate solution.
Whole livers remain unchanged in volume in a 3% solution, but
are shrunken by subsequent dehydration and retain only 49% of
their original volume when brought into paraffin wax. Primary
spermatocytes of the snail are reduced to 23% of their original
volume when paraffin sections of the ovotestis fixed in a 5 % solu-
tion have been mounted in Canada balsam. This represents greater
final shrinkage than that which follows fixation by most primary
fixatives.

Hardening. Tissues are left very soft. Wetzel's figure for rigidity
after fixation in a 3% solution is 171. Chromium trioxide leaves
tissues 2*7 times as rigid as this. After subsequent soaking in 80%
ethanol tissues are still very soft.

In the old days, when potassium dichromate was used as a
hardening agent before sectioning by hand without embedding,
tissues were left in the solution for long periods.

Immediate effects on particular constituents of the cell. The shape
of the cell is rather well preserved, though there may be some
retraction of small pseudopodia. The ground cytoplasm becomes
somewhat granular. Mitochondria are preserved, but transformed
from threads into ovoids and short rods: their form would prob-
ably be better maintained in the presence of an indifferent salt.
Lipid globules tend to run together. The nucleus retains its form-
but may be somewhat retracted away from the cytoplasm; its

132 FIXATION

membrane is very clearly seen; the nuclear sap is finely granular;
the nucleolus is shrunken.

Washing out. An insoluble precipitate (presumably of chromic
oxide, CraOg) tends to be formed in tissues if they are transferred
directly from potassium dichromate to aqueous ethanol. It has
already been mentioned that the salt is insoluble in absolute
ethanol. Potassium dichromate is therefore usually washed out in
running water. The experiments of Virchow ^^^ suggest that it
may be safe to transfer tissues directly from potassium dichromate
solution to 95% alcohol if light be excluded. Overton ^"^ advised
washing tissues in sulphurous acid after fixation in potassium
dichromate (see p. 109).

Effect on dyeing. Seki *^^ claims that potassium dichromate
renders proteins and cytoplasm acidophil, but in fact cytoplasm
can be coloured quite strongly by certain basic dyes after the
action of this fixative. Chromatin is left strongly colourable by
basic dyes,^^ but it is not fixed in its original position within the
cell. Since the nuclear membrane is well fixed, the chromatin
cannot escape, but distributes itself almost at random within the
nucleus. This is the last fixative one would choose for studies of
chromosomes (unless acidified).

Effects on the histological picture seen in paraffin sections. It was
on paraffin sections of root- tips of maize {Zea mays) that the Ameri-
can cytologist Zirkle first clearly demonstrated the effect of pH on
fixation by potassium dichromate. He showed that when the
hydronium-ion concentration was on the more acid side of a certain
range, the fixation-image was that of chromium trioxide; on the
less acid side the image was completely different. Simple solutions
of potassium dichromate fall on the less acid side. Zirkle put the
change-over range at pH 4-2 to 5-2, but Casselman, in a recent
careful study with mammalian tissues, put it at pH 3-4 to 3-8.

By itself, potassium dichromate is a very poor fixative for paraf-
fin sections (grade V). Cellular aggregates shrink apart from one
another, leaving wide artificial spaces; cytoplasm is rather homo-
geneously fixed, but tends to shrink round the nuclei and some-
times even disintegrates partially, so that cells become separated
from one another; mitochondria are retained, though often some-
what rounded up ; red blood-corpuscles are swollen and irregular.
The shape of nuclei is fairly well retained ; the nuclear sap is homo-
geneously fixed without net-like coagulum, but may retract from
the membrane ; the nucleolus is shrunken and often surrounded by

PRIMARY fixatives: NON-COAGULANTS 133

a halo, and may be subdivided; the heterochromatic segments of
the chromosomes are not seen in the interphase nucleus, and the
definitive chromosomes of mitosis and meiosis are unfixed.

In brief summary and at some risk of over-simplification one
may express the effect of pH on the action of potassium dichromate
thus. On the less acid side of the critical range (pH 3-4 to 3-8), the
cytoplasm, nuclear sap, and mitotic spindle are homogeneously
fixed ; mitochondria are retained ; the nucleolus is partly dissolved ;
chromosomes are scarcely visible. On the more acid side potassium
dichromate acts like chromium trioxide: that is to say, cytoplasm
and nuclear sap are coarsely coagulated and the mitotic spindle is
fibrous; mitochondria are non-existent; the nucleolus and chromo-
somes are well fixed. These may be called respectively the less
acid and more acid fixation-images of the chrome anions, as seen
in paraffin sections.

Compatibility uitli other fixatives. Potassium dichromate is
compatible with picric acid, mercuric chloride, and osmium
tetroxide. If mixed with more than a very small amount of
chromium trioxide, it shows the more acid fixation-image. ^^^' ^^^
It also shows this image if mixed with more than a very small
amount of acetic acid or any other acid used in fixation. It reacts
rather slowly with formaldehyde, and mixture with this substance
is allowable if it is done immediately before use ; when the colour
changes, the fluid should be renewed. Potassium dichromate
solution should not be mixed with ethanol, lest chromic oxide be
deposited in the tissue.

Uticlassified remarks. It has been known since the end of the last
century that different dichromates give different fixation-images,^^
but the explanation awaited the work of Zirkle.^^^ The subject has
now been re-investigated by Casselman. ^^^ Those dichromates
that show, at fixative concentration, a pH on the more acid side of
the critical range, fix like chromium trioxide: barium, calcium,
mercuric, and silver dichromates are examples. A saturated solu-
tion of mercuric dichromate is particularly strongly acid (pH
1-05). Those that show a pH on the less acid side fix like potassium
dichromate. The ammonium, lithium, and sodium salts do this.
The last-named is the least acid, a solution of the same mole-
cular concentration as 2-5% potassium dichromate (pH 4*05)
showing a pH of 5-10.^^^ Ammonium dichromate presents the
advantage that it does not swell mitochondria, as the potassium
salt does.^^^

134

FIXATION

ACETIC ACID

Standard concentration for fixation. 5% v/v aqueous solution.

^°

Formida and formula-weight. H3C.C/ . 6o-o.

Description. Acetic acid is a colourless liquid with a pungent
smell. It boils at US'" C; the crystals formed on cooling melt at
1 6-6° C. The ease with which it may be frozen has given rise to the
name of 'glacial' acetic acid, which is applicable only when the
acid is free of water. The acid is miscible with water and ethanol
in all proportions.

Beyond its use as vinegar and for pickling, acetic acid is impor-
tant in the production of cellulose acetate plastics.

Ionization. Acetic is a moderately weak acid, with ionization
constant i-8o x 10"^. The pH of the 5% solution is 2-32 ^^^ (Seki^^^
gives 2-4).

Oxidation-potential. Acetic acid can act as an oxidizer on reduc-
tion to acetaldehyde, or oxidized by strong oxidizers to carbon
dioxide and water. The oxidation-potential of the 5% solution
is 077 volt.^^^

Manufacture. The best culinary vinegar is made by the oxidation
of the ethanol in wine or other alcoholic liquors through the
action of bacteria (generally Acetohacter spp.). Acetic acid is also
made by the destructive distillation of the sawdust of beech and
other hard-woods under a pressure of several atmospheres. The
distillate is wood-vinegar or pyroligneous acid, which contains
acetic acid at about 5%, with creosote and other contaminants. The
weak acetic acid made by either of these processes may be treated
with lime or soda to make calcium or sodium acetate. The salt is
distilled with sulphuric acid, and the distillate fractionally distilled
to give the glacial acid.

Acetic acid is also made by the oxidation of acetaldehyde with
atmospheric oxygen in the presence of cobalt acetate as catalyst;
the acetaldehyde is prepared from acetylene.

Introduction as fixative. Vinegar appears to have been used for
pickling vegetables from remote times, and it is remarkable that the
regular use of acetic acid in microtechnique is scarcely more than
a hundred years old. In the eighteenth century Henry Baker ^^ had
tried vinegar as a preservative for hydra, but without much success.
Quekett, in his book published more than a century later, ^^^ does

PRIMARY fixatives: NON-COAGULANTS 135

not even mention acetic acid. The softness of tissues fixed with
this substance probably counted against it, for the microscopists
of the time were more interested in hardening agents than in
fixatives. Corti,-^-^ who experimented freely with fixative fluids,
tried it in his study of the inner ear in 1851, and in the same year
Clarke ^^^ used it in a mixture with ethanol for the treatment of
tissues that had already been soaked in the latter fluid. It was
subsequently used by Remak ^^^ in 1854 and Auerbach ^^ 20 years
later. Flemming ^^^ mentioned that it made the nuclear membrane
very refractive and tended to distort it: he preferred chromium
trioxide and picric acid. Acetic acid appears to have been valued
in the seventies and eighties chiefly for showing nuclei clearly and
making connective tissue transparent; pyroligneous acid was
sometimes preferred, because it hardens somewhat. ^"^

Reactions with proteins. Acetic acid (at the standard concentra-
tion) does not coagulate albumin, does not set egg-white into a gel,
and has no fixative effect on gelatine/albumin gel or on haemoglo-
bin. Histone can be extracted from tissues by acetic acid. Its most
evident effects are to swell protein gels and fibres and to produce
a precipitate with nucleoprotein.

The undissociated acid is thought to break the linkages between
amide groups of contiguous protein chains, by associating with
these: this would permit swelling. The dissociated acid splits the
salt-links (amino to carboxyl) that also hold protein chains together,
and this again permits swelling. Water is drawn into the protein
by attraction to the hydrophil groups exposed by these reactions.
The hydronium ion has a preservative effect, because it checks
autolysis and stops the growth of putrefactive bacteria.

Acetic acid gives a thick precipitate with nucleoprotein solution.
This is attributed to the action of the acetate ion in splitting off
DNA from the protein. DNA is precipitated from solution by
acetic acid.

Reactions with lipids. Certain lipids are miscible with glacial
acetic acid, or soluble in it: sphingomyelin, the ricinolein of castor
oil, and cholesterol are examples (though the latter is only
slightly soluble). These facts, however, are not of much significance
for microtechnique, since lipids are not ordinarily soluble in
acetic acid at the usual fixative concentration of 5% or thereabouts.
Phospholipids can form colloidal solutions in water, but their
solubility in acetic acid of fixative strength does not appear to have
been determined. Acetic acid is not known to fix any lipid.

136 FIXATION

Reactions zvith carbohydrates. Acetic acid neither fixes nor
destroys carbohydrates.

Rate of penetration. Acetic acid penetrates at moderate speed into
gelatine/nucleoprotein gel (K = 2-75). It may be remembered that
its rate of penetration into gelatine/albumin gel cannot be measured,
because it does not fix this gel. For the same reason the rate of its
penetration into tissues cannot be measured in such a way as to
give a K- value comparable with the others quoted in this book (see
under potassium dichromate, p. 131). No doubt it runs quickly
through the tissues, as Tellyesniczky's data suggest (K = 1-2), but
it penetrates without fixing proteins, precipitating nucleic acids as
it goes.

Shrinkage or swelling. Acetic acid swells protein gels far more
than any other fixative, for reasons that have been discussed (p.
64). A simple aqueous gelatine gel (15% w/W), placed in acetic
acid solution, expands to about 13 times its original volume in a
week. Under the standard conditions of measurement, gelatine/
albumin gel expands to 455% of its original volume (see fig. i, p.
36). Tissues and cells also swell in acetic acid, but if they are not
stabilized in the swollen state by the action of some other fixative,
they shrink strongly on dehydration and subsequent treatment.
Thus the spermatocytes of the snail retain only 28% of their
original volume when paraffin sections have been mounted in
Canada balsam. They retain a considerably larger volume if
formaldehyde be used as fixative, though this causes very much less
initial swelling of protein gels.

Hardening. Acetic acid leaves tissues much softer than any other
fixative. Wetzel's figure for rigidity is only about 9. The figure for
chromium trioxide is 25 times as great. After subsequent soaking
in 80% alcohol, tissues remain extremely soft.

Immediate effects on particidar constituents of the cell. The cell-
outline becomes rather indistinct; any thin pseudopodia tend to be
transformed into rows of globules; ground cytoplasm loses its
original homogeneity; mitochondria are transformed into faint
rows of granules ^^^' *^^ and generally disappear; lipid globules
are sometimes well retained, but the neutrophil granules of
polymorphs disappear. The nucleus sometimes retracts from
the cytoplasm; the nuclear contents are transformed into a
lumpy network; the nucleolus sometimes becomes irregular in
shape.

Methods of washing out. Since acetic acid is perfectly miscible

PRIMARY fixatives: NON- COAGULANTS 137

with ethanol and has no tendency to produce insoluble extrinsic
artifacts, no special washing out is necessary.

Ejfect on dyeing. Cytoplasm is rendered rather strongly acido-
phil, though it will also take basic dyes. The chromatin of inter-
phase nuclei colours rather feebly with basic dyes, and scarcely at
all with acid ones (probably because it is represented only by DNA,
the protein constituent having dissolved away). Metaphase and
anaphase chromosomes colour strongly with basic dyes. The
nucleolus is not readily coloured by dye-lakes.

Effects on the histological picture seen in paraffin sections. Zirkle ^^^
showed that the acetic anion only produced its characteristic
fixation image if used on the acid side of pH 4.0 or thereabouts. At
less acid pH than this, fixation does not occur and tissues macerate:
little beyond the nucleoli can be identified in paraffin sections of
the macerated material.

The typical 'acid' fixation-image may be summarized thus.
Cell-aggregates tend to be widely separated from one another by
artificial spaces. Cytoplasm is poorly represented: it is strongly
contracted round the nuclei, or coarsely reticular. Mitochondria
are not seen: this is particularly characteristic of acetic fixation.
The shape of nuclei is fairly well retained, but the nuclear sap
seems not to be fixed and there is only a coarse reticulum within
the interphase nucleus, with a swollen, often vacuolate nucleolus.
Definitive chromosomes are rather well fixed. The mitotic spindle
appears fibrous.

Zirkle ^^^ considered that the fixation-image was similar to that
given by chromium trioxide, and there are indeed similarities.
Nevertheless, chromium trioxide (with sodium chloride) gives
better general fixation. The cellular aggregates stand in more life-
like relation to one another; chromosomes at all stages are better
fixed ; the nucleolus retains its original form.

It is not obvious why mitochondria do not appear in paraffin
sections fixed with acetic acid alone. Their lipid content has not
been proved to be soluble in the 5% solution, and indeed, as we
have seen, they are not necessarily destroyed by the action of the
fixative itself, though as a rule they are either destroyed in the
fixative or else left in a condition that results in their destruction at
a subsequent stage. In a few cases they can be seen in paraffin
sections of material fixed in mixtures containing a considerable
amount of acetic acid.^^ Despite the general belief to the contrary,
it must be the acetate ion or the undissociated acid that acts

138 FIXATION

unfavourably upon them, not the hydronium ion ; for as Casselman
and Jordan ^^* showed, mitochondria can be quite well seen in
paraffin sections of tissues fixed in o-iN hydrochloric acid.

Compatihility with other fixatives. Acetic acid is compatible with
all other fixatives, but when it is mixed with potassium dichromate,
the fixation-image of chromium trioxide is given.

Unclassified remarks. The fixation- image given by acetates in
paraffin sections is dependent mainly on their pH. This was
shown by Zirkle,^^'^ who experimented with various salts con-
taining the same amount of the acetate ion as 2% acetic acid.
Those acetates that gave a pH less than 4-0 (bismuth subacetate,
for instance) tended to produce the characteristic 'acid' fixation-
image of acetic acid. Sodium acetate, on the contrary, and others
that also gave a less acid pH than 4-0, generally macerated tissues.
In some cases the cation affected the image.

The other short-chain fatty acids (formic, propionic, butyric,
valeric) all give much the same fixation-image in paraffin sections
as acetic; so do glycollic, glyceric, lactic, and gluconic. ^^^ Tri-
chloracetic acid, however, acts in an entirely different way. It
is a coagulant fixative; it leaves the nucleolus readily colourable
by iron haematein, and mitochondria can be well fixed by mix-
tures containing it at 2%.^^^' ^^^

CHAPTER 7

Fixative Mixtures

The term 'fixative mixtures' is here used to mean mixtures of
two or more substances, each of which acts as a fixative when used
alone. A primary or unmixed fixative is regarded as remaining
primary when nothing but an indifferent salt or other non-
fixative substance is added to it.

The primary fixatives present the advantage that the inter-
pretation of their effects — difficult enough though it may be — is
much easier than that of mixtures. Still, the majority of successful
fixatives used in routine work are mixtures. Ethanol is a poor
fixative (grade IV), acetic acid an indifferent one (grade III): but
mixed together in appropriate proportions in Clarke's fluid ^'-'' ^^^
they produce a fixative that is not only very good (grade I) in
routine histology, but also valuable in chromosome studies.

Most of the mixtures used today have come into being in a
haphazard way. A study of the papers in which the formulae
were first published will show this. One expects to find a careful
consideration of the causes that led the author to choose certain
primary fixatives and to mix them in particular proportions, but
usually nothing of the kind is offered: the mixture is presented to
the reader as a. fait accompli, quite frequently in the form of a foot-
note. Occasionally the author tells us about the various mixtures
he tried empirically, but the description of his experiments shows
that he gave no consideration to the fact that the ingredients must
necessarily interact.

It is clear that a process of natural selection has been at work.
Many new mixtures have been thrown up more or less at random
by processes analogous to mutation and recombination, and they
have been tried out in practice by a number of independent workers.
Only the ones that give reasonably good results continue to be
used subsequently: many fall by the wayside in the struggle for
existence, or drag out a futile old age in the pages of the recipe-
books.

139

140 FIXATION

Some authors delight in making trivial changes in well-known
formulae. Champy's fluid, ^^^ for instance, has this composition: —

potassium dichromate, 3% aq. . . . 7 ml

chromium trioxide, 1% aq. . . . 7 ml

osmium tetroxide, 2% aq. . . • . . 4 ml

This is a useful fixative for certain cytoplasmic inclusions.
Nassonov ^®* used the proportions 4:4:2 (he calls it Champy's
fluid, without comment). To this he adds a solution of pyrogallol,
measured in drops: the total amount of pyrogallol added is about
o-i mg to 10 ml of fluid. Pyrogallol could not exist for an instant
in the presence of vastly greater amounts of two very strong oxi-
dizers (chromium trioxide and acidified potassium dichromate) and
one moderately strong one (osmium tetroxide): it must at once be
changed to carbon dioxide and other oxidation-products. Yet
many cytologists continue to believe that there is some special
virtue in Nassonov's fluid. Actually there is none. This can be
proved by getting a friend to fix one set of objects in Champy's
fluid and another in Nassonov's, with secrecy as to which is which.
It will not be found possible to distinguish the final preparations.

Fixatives are generally named after the persons w^ho invented
them. It has already been mentioned (p. 24) that it is often con-
venient to call them simply by the names of the inventors, without
necessarily saying So-and-so's fluid. This works well when the
inventor (Zenker, for instance) only introduces a single fixative.
When someone introduces two or more, descriptive words are
necessary. Thus one may refer to Flemming's weak ^'^^ and
strong ^"^ mixtures. It is desirable in such cases that the inventor
should himself suggest suitable names. Heidenhain ^^^ named one
of his fluids Susa, combining into a single word the first two letters
of each of the words Suhlimat and Sdiire. (Some authors have
supposed Susa to be a person.) It is thoughtless of an inventor to
call a fluid by the number that it happens to receive in his labora-
tory note-book ('B.15', for instance, or '2BD'), for this has no
mnemonic value for others.

In some cases the reduction or omission of one constituent
radically changes the nature of a fixative, and a change of name is
then desirable. Flemming's strong fluid ^"^ contains i Maastheil
oder weniger of acetic acid to 19 of other constituents. Benda ^^
reduced the amount to 3 drops of acetic to 19 ml of other constitu-
ents: Lewitsky ^^^ omitted the acetic acid altogether. Flemming's

FIXATIVE MIXTURES I41

fluid, as usually used, is a valuable fixative for the study of chromo-
somes and for the detailed histology of very small pieces ; those of
Benda and Lewitsky are quite different in character, being in-
tended for work on cytoplasmic inclusions, and should be called
by the names of the men who introduced them.

Fixative mixtures are not always ascribed to their actual in-
ventors. Thus Clarke ^^'^ introduced in 1851 a mixture of one vol-
ume of acetic acid with three of spirits of wine ; in this he soaked
tissues that had already been immersed in spirits of wine alone.
This mixture was widely used as a direct fixative in the following
years, by Beale ^^ and others. Frey ^"^ quoted it in his well-
known text-book in 1863, giving die Clarke' sche Vorschrift as
3 Theile Alkohol niit i Theil Essigsdure. He continued to quote it
repeatedly in his various editions. ^"^ It is therefore rather strange
that the mixture should nowadays be almost invariably attributed
to Carnoy,^^^ who gave this formula in 1886. It will here be called
by Clarke's name, while Carnoy's will stand for the fluid of his
own invention ^^^ (absolute ethanol, glacial acetic acid, and
chloroform in the proportion of 6 : i : 3 by volume). It may be
remarked that Clarke is the better fixative for routine paraffin
sections: it falls in grade I, Carnoy in grade II.

Several authors have mixed a saturated solution of mercuric
chloride with glacial acetic acid, but it does not seem possible to
find out who first used the familiar mixture of the two substances
in the proportion of 95 : 5 by volume. This fluid (here called
mercuric/acetic) is useful in zoology, particularly in the preparation
of whole mounts.

Another anonymous mixture is Zenker without acetic. The fluid
is radically different from Zenker, because the pH lies on the
opposite side of the critical range (p. 132), and proteins therefore
react to the chrome anions in it in an entirely different way. This
is the only fixative that is useful in cytoplasmic cytology and at the
same time good enough for routine histology to reach grade I.
Bensley ^^ has recommended a fluid closely similar to Zenker
without acetic, but not identical with it.

For the purpose of generalization it is necessary to choose a
limited number of representative fluids. Twenty-five aqueous
fixative mixtures have been selected for this purpose, and two not
containing water. They are a typical selection of mixtures that are
widely used in micro-anatomy, embryology, histology, and cyto-
logy. Different authors would have made different lists, but a

142

FIXATION

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144 FIXATION

considerable proportion of the 27 would almost inevitably have
been chosen. The aqueous mixtures are listed in table 8. The two
non-aqueous ones are Clarke and Carnoy.

The dates of introduction of these 27 fixatives have some in-
terest from the historical point of view. The dates of invention of
two are unknown. The formulae for the remaining 25 were first
published in the several decades as follows: — •

1850-59 I

1860-69 o

1 870-79 I

1880-89 4

1890-99 6

1900-09 4

1910-19 7

1920-29 2

If the list of fixative mixtures commonly used today were con-
siderably extended, it would probably be found that the great
majority of them were invented between 1880 and 1919. The most
ancient fixative mixture commonly used today is Clarke (1851).^'-"

Table 8 gives various kinds of numerical information about the
25 aqueous fixatives. It was found impossible to make a direct com-
parison between all the fixatives in the table except by relating the
amount of each ingredient to that of the only substance that
occurs in all: namely, water. The concentrations are therefore
expressed as percentages, w/W, except that acetic acid is expressed
as v/W, since this substance is nearly always measured by volume
in biological laboratories. (See Appendix, p. 313.)

Bouin's fluid ^^ may be used as an example to explain how the
concentrations were calculated. The composition is: —

formalin . . . . . . 25 ml

picric acid, sat. aq. . . . . . 75 ml

glacial acetic acid . . . . . 5 ml

The approximate composition of the formalin is 10 g of form-
aldehyde and 15 g of water. The water in the picric acid solution
is very nearly 75 g. The total amount of water may therefore be
taken as 15 + 75 = 90 g. Picric acid is soluble at about 1*4% in
water: thus there is about 1-05 g in 75 ml of the saturated solution.
The composition of the fluid is therefore: —

FIXATIVE MIXTURES

H5

formaldehyde
picric acid .
acetic acid (glacial)
water .

10 g

1*05 g
5 nil
90 g

If each of these figures be increased by one-ninth, we have the
composition expressed as percentages of the weight of w^ater: —

formaldehyde . . . . , 11 g

picric acid . . . . . . i-2 g

acetic acid (glacial) .... 5-6 ml.

The concentrations of the solutes are therefore: — formaldehyde,
11% w/W; picric acid, i-2%, w/W; acetic acid, 5-6% v/W. These
are the figures given in table 8.

Care has been taken to make the table as accurate as possible,
but perfect accuracy cannot be achieved. Some inventors of
fixative mixtures give amounts in drops. It is impossible to be sure
how much HXO3 there w^as in Perenyi's Saltpetersdure 10%.
Saturated solutions are of varying concentrations. Authors do not
tell us how much (if any) water of crystallization they introduce

TABLE 9

Analysis of 2$ aqueous fixative mixtures: summary of part of the
information contained in table 8.

No. of

Concentration,

'

/o

mixtures
in which
each occurs

Minimum

Mean

Maxitnum

ethanol

3

6-5

76-5

180

picric acid

4

0-35

i-o

1-4

mercuric chloride .

8

2-2

4-8

6-8

chromium trioxide .

8

0-2I

0-73

17

formaldehyde

II

1-4

9-8

36

osmium tetroxide

9

0-I7

0-47

i-o

potassium dichromate,

scarcely or not acidified

7

2-0

2-4

2-6

potassium dichromate,

acidified

2

1-2

1-8

2-5

acetic acid

13

0-I2

5 '2

23

with sodium sulphate. The attempt has been made to get the
concentrations correct, where possible, to two significant figures.
There w^ould be no point in trying to go beyond this.

The table will enable the reader to make a direct comparison of
any particular fixative mixture in the list with any other. Certain

K

146 FIXATION

general conclusions can also be drawn. Some of these are shown in
table 9. It will be noticed what a preponderating place is taken in
our most familiar mixtures by the eight primary fixatives described
in detail in chapters 5 and 6. The only other fixative substances
that occur in any of these 25 mixtures are nitric acid, sulphuric
acid, chloroplatinic acid, trichloracetic acid, cadmium chloride,
and creosote. Of these, nitric acid occurs in two of the 25 mixtures,
each of the rest in one only. It will be remembered that the two
representative non-aqueous mixtures (Clarke and Carnoy) both
contain ethanol and acetic acid ; these substances therefore appear
respectively in 5 and 15 of the 27 chosen mixtures.

The two occurrences of acidified potassium dichromate should
properly be added to the 8 of chromium trioxide, since, as we
have seen (p. 127), the ions are essentially the same.

The pH range of the 25 aqueous mixtures extends only from
0*3 to 4-9. It may be remembered that one of the primary fixatives
(formaldehyde) is sometimes used in alkaline solution (p. 112).

It is not the purpose of table 8 to help in the making up of the
various mixtures. It is best to keep stock solutions of most of the
primary fixatives at suitable concentrations and to make up mix-
tures from these. In cytological work one requires very small
amounts of fixatives. If a piece of tissue is only about a millimetre
in greatest diameter, 2 ml of fixative are amply sufficient: 4 ml are
enough for nearly all cytological purposes. It is convenient to
have stock solutions from which several useful fixatives can be
made up. Three valuable mixtures are Hermann, Flemming's
strong fluid with full acetic, and a fixative called Lewitsky-saline
(LS). The latter is Lewitsky's fiuid with the addition of sodium
chloride: it can be strongly recommended for studies of cyto-
plasmic inclusions.^^ These three fixatives can be made up from
stock solutions in the way shown in table 10. If these small amounts
of fixatives are used, the high cost of osmium tetroxide and chloro-
platinic acid no longer forbids their frequent use. Flemming's
fluid is not stable (though it does not change colour), for chromium
trioxide slowly oxidizes acetic acid; it is therefore always best to
make up very small amounts.

Quite a number of commonly used mixtures are best made up
at the moment of use, since they contain ingredients that interact.
Some part of the mixture may generally, however, be made, and
the fixative will then be available at short notice. Thus for
Sanfelice's fluid the chromium trioxide must be kept separate

FIXATIVE MIXTURES 147

from the formaldehyde till the last moment, but there is no reason
why the formaldehyde should not be mixed with the acetic acid.
Although certain very unstable fixatives w^ork well, yet it is
questionable whether they could not be replaced by more rational
mixtures. Some of the changes that occur in unstable fixatives have
been studied by Freeman and his colleagues ^"^ and by Cassel-
man.^^^ The fluids of Helly, Karpechenko, Orth, and Regaud all

TABLE 10

Method for making up small quantities of certain cytologic al fixatiz'es.
It is often convenient to halve these quantities.

Flemming ^ '^

Hermann ^^^

Lezvitsky-

saline ^^

ml

{strong, full

{mammalian

acetic)

formula)

ml

ml

Distilled water .

1-6

1-6

2-0

Chromium trioxide, 5% aq.

0-6

0-6

Chloroplatinic acid, 5% aq. .

0-6

—

Sodium chloride, 5% aq.

0-6

Osmium tetroxide, 2% aq. .

0-8

0-8

0-8

Acetic acid, 20% aq. .

i-o

i-o

rise gradually in pH from the time when they are made up. Thus
Helly rises from pH 3-70 when fresh to pH 4-20 on the next day.
The oxidation-potential falls at the same time; that of Sanfelice
falls even more. Other fixative mixtures, such as Altmann, Bouin,
Champy, Susa, Hermann, Perenyi, and Zenker, maintain a con-
stant or nearly constant pH, and at least some of them (Altmann,
Champy, Zenker) a constant oxidation-potential; Flemming with
full acetic becomes more acid on standing.

The products of the reactions in unstable mixtures appear not
to have been worked out. The reduction of the chrome anions is
likely to produce cationic chromium; this would itself act as a
fixative, though quite differently from the anions. The use of
cationic chromium in fixation has been suggested by Zirkle.^^^ If
unstable mixtures are really necessary, it is important that their
reaction-products and the effects of these on the tissues should be
known.

Of the fixatives listed in table 8, nearly all that are used chiefly
in micro-anatomy, embryology, histolog}^ and in studies of
chromosomes contain acetic acid. This applies also to Clarke and
Carnoy. The only exceptions are Kleinenberg (which contains
sulphuric acid) and Helly. For chromosome studies acetic acid is

148 FIXATION

almost invariably included, though exceptions can be quoted.^*®' ^^^
Fixatives for chromosomes usually also contain chromium trioxide
(or acidified potassium dichromate).

Many of the mixtures used for the purposes mentioned in the
last paragraph contain, in addition to acetic acid, one or more
coagulant fixatives and either formaldehyde or osmium tetroxide.
This applies, for instance, to Allen's B.15, Bouin, Brasil, Flem-
ming, Susa, Hermann, Karpechenko-Navaschin, and Sanfelice.
This trio of ingredients (acetic acid + coagulant + non-coagulant
fixative of ground cytoplasm) is so frequent in widely-used mix-
tures that the reasons for its success must be sought. Acetic acid
does not in itself prevent great shrinkage in final preparations
(p. 136), but it does prevent it during fixation. In mixtures it
presumably antagonizes the shrinking effect of other fixatives
while the latter are stabilizing the proteins in the unshrunken
state; it also prevents excessive hardening. Ross ^^^ showed that
mixtures have much less tendency than primary fixatives have to
produce a badly-shrunk final preparation. The primary spermato-
cytes of the snail, fixed in 4% formaldehyde, retain in balsam only
34% of their original volume, and this appears to be the primary
fixative that results in the least final shrinkage of any (with the
doubtful exception of osmium tetroxide). With chromium tri-
oxide at 0-75% the figure is 29% of the original volume; with 5%
acetic acid, 28%. Yet when these three primary fixatives are mixed
to produce Sanfelice, the final volume in the mounted paraffin
section is 66% of the original. Sanfelice gives less final shrinkage
than any other mixture studied by Ross. All the mixtures he
studied (including Bouin, Helly, strong Flemming, mercuric/
acetic, and Zenker) gave less final shrinkage than any of the pri-
mary fixatives (except perhaps osmium tetroxide).

This only explains in part why fixatives containing the trio are
so successful. Why should not acetic acid and one other ingredient
suffice? It seems that tissues are not readily infiltrated with paraffin
unless they have been made spongy to some extent by the inclusion
of a coagulant fixative. If, however, a coagulant is used alone, the
cytoplasm and nuclear sap tend to be transformed into rather
coarse meshworks. If formaldehyde or osmium tetroxide be in-
cluded, a compromise is reached: the protoplasm is more smoothly
fixed, but paraffin can still enter.

The requirements of paraffin embedding have in the past to a
large extent controlled our choice of fixatives. The introduction

FIXATIVE MIXTURES 149

of new embedding media is likely to result in less reliance being
placed on coagulant fixatives.

It follows from what was said in chapter 6 that fixative mixtures
intended for the study of cytoplasmic inclusions will contain
formaldehyde, osmium tetroxide, or unacidified potassium
dichromate, or more than one of these. Acetic acid is omitted or
reduced to a minute amount. These generalizations apply to Alt-
mann, Aoyama, Benda, Bensley's AOB, Champy, Helly, Lewitsky,
Mann, Orth, Regaud, and Zenker without acetic. Of these, only
Benda and AOB contain acetic acid, at great dilution (less than
0-2%). It must be emphasized once more that the omission or
great reduction of acetic acid is not necessarily connected with pH.
Thus Lewitsky differs from Flemming's strong fluid solely in the
absence of acetic acid from the former: yet the two have the same
pH.^^^ Lewitsky's fluid was designed specifically to fix mito-
chondria, while Flemming (with full acetic) destroys them. This
shows the specific destructive (or at any rate non-fixative) effect of
acetic acid on mitochondria. This effect, as we have seen (p. 138),
is not shared by hydrochloric acid. Acetic or any other acid, unless
very dilute, will transform potassium dichromate into a coagulant
fixative and in so doing abolish its ability to fix mitochondria.

Coagulants are often omitted from mixtures used for the study
of cytoplasmic inclusions. This applies, for instance, to Altmann,
AOB, Orth, and Regaud. Easy infiltration with paraffin is sacri-
ficed for the sake of homogeneous fixation of the ground cyto-
plasm. It is particularly difficult to obtain good paraffin sections of
tissues fixed in Altmann.

Each component of a fixative mixture should so far as possible
compensate for a defect in another. Thus ethanol shrinks tissues
strongly and does not fix chromatin: acetic acid compensates for
both these defects. Acetic acid does not fix cytoplasm or nuclear
sap: ethanol fixes both (though the former indifferently). When
ethanol and acetic acid are mixed to make Clarke's fluid, an
excellent fixative for routine histolog}^ and chromosome studies is
produced.

The properties of Bouin's fluid can be analysed in a similar way.
Formaldehyde fixes cytoplasm and nuclear sap, but hardens
tissues unduly, prevents paraffin from penetrating easily, makes
cytoplasm basiphil (so that acid dyes do not work well), and fixes
definitive chromosomes poorly. Picric acid compensates for most of
these defects. It leaves tissues soft, coagulates cytoplasm in such a

150 FIXATION

way that it readily admits paraffin, makes it strongly acidophil,
and fixes chromosomes rather well. It has, however, two serious
defects: it shrinks tissues badly and makes chromatin acidophil.
Acetic acid compensates for both these defects.

Unfortunately the good qualities of primary fixatives cannot
always be combined in mixtures. Potassium dichromate tends to
stabilize the cytoplasm and nuclear sap in a homogeneous state,
but dissolves and disperses chromatin: acetic acid fixes chromatin
but does not fix cytoplasm or nuclear sap. The attempt was
naturally made to let each compensate for the defects of the other.
Yet Tellyesniczky's fluid *^^ did not and could not achieve its
objects, because the acidification of the chrome anions causes
them to act as though chromium trioxide had been dissolved
instead of potassium dichromate, and this is a strongly coagulative
fixative that does not stabilize cytoplasm or nuclear sap as a
homogeneous gel.

In deciding which primary fixatives to mix, it is important to
take into account not only their obvious mutual compatibilities and
incompatibilities, but also their more subtle influences on one
another's properties. Thus anyone who includes mercuric chloride
in a mixture should remember that its coagulative power and the
solubility of its coagulates are affected by acidification (p. 52).

The effect of so-called indifferent salts must also be con-
sidered. Sodium chloride, as we have seen (p. 54), can dissolve
protein coagulates produced by mercuric chloride, in certain
circumstances. Ammonium sulphate can transform ferric sulphate,
at certain concentrations, from a non-coagulant to a coagulant
fixative (p. 85). Indifferent salts can be very useful ingredients of
mixtures, but it is unlikely that they improve any fixative that
contains acetic acid at 5% or thereabouts.

All the fixative ingredients of an ideal mixture would penetrate
at the same speed. This could be achieved by adjustment of their
concentrations. It does not appear that any inventor of a fixative
mixture has taken this into consideration. A section through a
large piece of tissue often gives unmistakeable evidence that one
ingredient reached the centre at fixative concentration in advance
of the others. When a small piece of tissue is used, the unevenness
due to this cause is minimized. The argument presented in detail
on p. 69 applies here. When a piece of tissue has been placed in the
fixative mixture, each ingredient must be supposed instantly to
reach the opposite side of it from that at which it started, at in-

FIXATIVE MIXTURES 15I

finitesimal concentration ; from then onwards it will help to build
up the concentration there faster than it would have been built up
if the fixative had come from one side only. It follows that
although perfectly even fixation by all the ingredients at the same
time could only be achieved if they all had the same K- value, yet
the smaller the piece of tissue, the more even the fixation will be.

PART TWO

DYEING

CHAPTER 8

Introduction to the Chemical
Composition oj Dyes

The constituent parts of cells and of intercellular material are
usually transparent and colourless, and therefore not distinguish-
able from one another unless there are appreciable differences of
refractive index. It was Leeuwenhoek (17 19) ^^^ who first used a
colouring agent in an attempt to overcome this difficulty. He was
studying mammaUan muscle, and tried the effect of an alcoholic
solution of saffron. ^^^ His experiment was not very successful, and
others wxre slow to follow his example. Iodine was used from time
to time for the same purpose, but during a very long period cells
and tissues were commonly examined in their natural, transparent
state. During the decade 1 848-1 858 the use of colouring agents in
microtechnique was repeatedly rediscovered,-^ and from that time
to the present day enormous use has been made of artificially
coloured preparations.

The colouring agents used in microtechnique are of diverse
kinds, but the majority of them are dyes. It will be best to consider
carefully what dyes are, before discussing the other kinds of
colouring agents that can be used (p. 296).

If a porous body, such as a sponge, be placed in a vessel con-
taining a solution of a coloured substance, some of the coloured
substance will be imbibed by the porous body, and if the latter be
lifted out of the solution, will be rem^oved with it. There are now
two possibilities about the solution that remains in the vessel.
Either the coloured substance is at the same concentration as it was
before the porous body was put in it, or else the concentration has
been reduced (that is to say, the solution has been to some extent
'exhausted'). If exhaustion has occurred, the porous body has
clearly shown a special affinity for the coloured substance in
preference to the solvent. It is characteristic of the process of dye-
ing that this special affinity exists, and indeed in the commercial

155

156 DYEING

dyeing of textiles (though not in microtechnique), exhaustion is
often nearly complete.

If, then, a substance acts as a dye, two problems present them-
selves: why is it coloured, and why is there an affinity between it
and the object that it dyes? The present chapter is concerned with
the first of these problems; the second will be considered in
chapter 10.

All dyes, in the strict sense of the word, are organic compounds.
It is a remarkable fact that very few aliphatic substances are
coloured. Aromatic substances, on the contrary, have a tendency
to absorb electro-magnetic weaves. Whenever we can write two or
more equally good structural formulae for a particular substance,
it is supposed that there is a possibility of rapid change in the
configuration of the molecule between the various possible states,
and this change or resonance involves the absorption of electro-
magnetic waves.

Alternative structural formulae for benzene

If our eyes were sensitive in the ultra-violet, benzene would
appear coloured, for it has an absorption-band at wave-length
256 m^Lt; and other aromatic compounds have absorption-bands at
various wave-lengths in the ultra-violet. There is a tendency for
resonance to be transmitted through a path of alternate double and
single valency-bonds. Indeed, aromatic compounds tend to absorb
electro-magnetic waves at particular w^ave-lengths just because
there is this alternation of bonds. Annatto (bixin), a plant-product
used in the colouring of butter and cheese, is of particular interest
in this connexion, for it is one of the rare aliphatic compounds
that are strongly coloured, and its long molecule presents that
alternation of double and single bonds that is so characteristic of
aromatic substances.

As a general rule, the alternation of bonds does not in itself
shake up the molecule sufficiently to extend the absorption-bands
into the visible region and thus produce colour. Any configuration
of the molecule that pushes the absorption bands downwards into
longer wave-lengths is said to be hathochromic^ and certain par-
ticular configurations are so effective in this respect that their
presence is always associated with colour. One of these is the
quinonoid arrangement. Quinone itself (parabenzoquinone) is

THE CHEMICAL COMPOSITION OF DYES 157

coloured (yellow), but it is chiefly when a quinonoid ring is intro-
duced into more complicated compounds that brilliant colours are
produced. Since the paraquinonoid ring confers the property of
colour, it is called a chromophore or colour-bearer. It occurs in
many dyes that are important in microtechnique. Other dyes owe
their colour to other chromophores, which will be mentioned in

chapter 9.

O

Parabenzoqiiinone

Quinone itself has no tendency to be exhausted from its solvent
by attaching itself to a textile or to the substance of a microscopical
preparation. In general, substances that have this tendency ionize
in aqueous solution, and quinone does not. A chromophore by
itself, then, does not confer upon a molecule the capacity of acting

II

The paraqiiinojioid ring

as a dye. An ionizing group is required as well. Those ionizing
groups that transform substances that are merely coloured into
dyes are called aiixochromes. As their name suggests, they have a
tendency to increase the intensity of the colour, often very
markedly, and it is sometimes convenient to think of the auxo-
chromophoric systems in dyes, in order not to distinguish too sharply
between the effects of chromophore and auxochrome.

One of the commonest auxochromes in dyes is the group -NHg,

Aniline

and it is because aniline contains this group that it is of such partic-
lar importance in dye-chemistry.

To make a dye, it is only necessary to combine a molecule that
can spring into a chromophoric configuration with another that

158 DYEING

contains an auxochrome. We may start with two colourless sub-
stances and produce a dye in a single process. A very easy one to
make is the dye known commercially 2is par arosaniline. (The par a-
in this name has no connexion with the chemical usage of the

NH, NH,

Aniline Aniline

CH3

NH2

Paratoluidine

prefix. It means that the dye is a modified form of another dye
with the commercial name of rosaniline.) Practical instructions
are given on p. 321. It is only necessary to bring together some
aniline and paratoluidine and to heat them in the presence of
chloride and a suitable oxidizing agent. The quinonoid chromo-
phore then appears, and the -NHg groups are already present to
act as auxochromes.

NH, NH,

NH2 cr

+
Pararosaniline

Pararosaniline is a solid, magenta substance, soluble in water
and alcohol.

In this book attention will be directed to quinonoid chromo-
phores by the drawing in of double bonds in quinonoid rings, but
aryl rings will be represented by a simple hexagons.

It will be noticed that one of the three rings — the quinonoid one
— has been shown with a positive charge on the nitrogen atom, a
charge balanced by the negative charge on the chloride ion. It
must not be supposed, however, that the position of this charge is
constant. On the contrary, it changes continually. It may be

THE CHEMICAL COMPOSITION OF DYES

159

associated with any of the three nitrogen atoms, or with the central
carbon atom, and indeed there are other possible positions. It is
the resonance between the particular positions that are available
in a molecule that determines the colour. This fact will be strik-

ci-

NH,

NH,

^

?'

NH2

Pararosaniline : another resonance position

ingly illustrated below, by reference to a related dye, crystal
violet (p. 168).

It is accurate to regard the positive charge as belonging to the
ion as a whole, and therefore to write the formula in the way
shown here.

NH, NH5

+

[CI]-

Pararosaniline : another formula

This kind of symbol will nevertheless not be used in this book,
because it is convenient to draw special attention to the chromo-
phore. The reader should remember that the formulae that will
be used represent, as it were, instantaneous photographs of the
dye-ion. He may decide to work out for himself the changes in the
position of the electric charge involved in the process of resonance.

The colour of the dye is very slightly changed if, instead of
taking t^vo molecules of aniline and one of paratoluidine, we take
one of aniline, one of orthotoluidine, and one of paratoluidine.
The dye formed from these constituents is called rosaniline. It will
be noticed that it differs from pararosaniline only in the possession
of the methyl group introduced with orthotoluidine.

l6o DYEING

The methyl group affects the colour, for rosaniline is a very
slightly bluer magenta than pararosaniline. Side-groups such as
this are called modifiers.

The familiar dye called basic fuchsine is a mixture of pararosani-
line with rosaniline; some specimens contain another related dye
as well. Since pararosaniline is the simplest of the triarylmethane
dyes, it could with advantage have been used as a type in the

NH2 NH2

,CH3

\
\

Y

NH2 ci-

+

Rosaniline

experiments described later in this book (p. 324); but whereas one
must make pararosaniline for oneself, basic fuchsine is readily
available in commerce, and for this reason it has been chosen
instead. Almost exactly the same results would be obtained if
pararosaniline had been used.

Magenta is another name for basic fuchsine. Mere inspection of
the solution suggests that it transmits much red light and some
blue, but absorbs light of intermediate wave-length. For accurate
comparison of the colours of dyes it is necessary to obtain figures
that will show the transmission or absorption at various wave-
lengths. This is done by use of a spectrophotometer. Light of
known wave-length is shone through a solution of the dye con-
tained in a flat-sided vessel of uniform thickness, and the intensity
of the light that comes through is measured by means of a photo-
electric cell. A measurement can also be made with exactly the
same apparatus and the same solvent (water, for instance), in the
absence of the dye. The amount of light that comes through when
the dye is present with the solvent can then be expressed as a
percentage of the amount that comes through when it is not. This
percentage is known as the transmission at the particular wave-
length used. It is usual to find the transmission at 10 mjx intervals
throughout the visible spectrum. A curve like that shown in fig. 17
is then obtained. It is generally best to dissolve the dye at a con-

THE CHEMICAL COMPOSITION OF DYES l6l

centration that will give a transmission of about 20% at the wave-
length of maximal absorption. It will be observed from the graph
that basic fuchsine transmits most of the red and orange light, and
also a considerable amount of violet and blue, but absorbs strongly
in the green, especially at about 540 m/x. Different specimens of
this mixed dye naturally differ somewhat in the w^ave-length of
greatest absorption.

100

80

60

o

m
in

to

z
<

40

20

400 450 500 550 600 650

WAVELENGTH, mp.

FIG. 17. Graph showing the transmission of light through a layer i cm
thick of basic fuchsine, o-ooo62°o aqueous. ^^ The following abbreviations
are used in this and subsequent figures: — v, violet; b, blue; g, green; y,

yellow; 0, orange; r, red.

Since the active property of the dye is to absorb, not to transmit
light, it is for some purposes desirable to express its absorptive
capacity as the reciprocal of the transmission ; for instance, instead
of saying that the transmission at a particular wave-length is y^
of the incident light, we may turn the fraction upside down and
use the number 5 to represent the absorption. A curve of re-
ciprocals for the same solution of basic fuchsine as before is shown
in fig. 18. Alternatively, one may use the logarithms of the re-
ciprocals. Such logarithms are called 'densities'. This method of
expression is particularly useful with light-filters, because if one
know^s the density of each at any w^ave-length, it is only necessary

L

1 62

DYEING

to add the two densities together to obtain the density of two
filters used one behind the other. As a result of this, the absorptive
capacity of coloured substances is often given in the form of
density-curves, such as that shown in fig. 19. It is necessary to
understand this method of representation, because it is so com-

5-

° 4

to

to

2

to

<

ct 3

<

o
o 2

O

UJ

QC

V
I

b
I

g
I

o

I

400

450

500 550

WAVELENGTH, m^

600

650

FIG. 18. Graph showing the reciprocals of the transmission of Hght
through a layer i cm thick of basic fuchsine, 0-00062*^0 aqueous. ^^

monly used; but in fact one seldom makes practical use of the
ease of addition of densities, if one's main interest is in the use of
dyes in microtechnique; and transmission gives a much more
direct statement of what is actually the important point when one
looks through the microscope — the colour of the dye.

Any change in structure that destroys the quinonoid linkage
results in the loss of colour. Many dyes can be changed to colour-
less or Z^wco-compounds by the action of reducing agents such as
sodium hydrosulphite (Na2S204). The leuco-representative of
pararosaniline may be regarded as a triaryl derivative of methane:
that is to say, as methane in w^hich three of the four hydrogen atoms
have been replaced by aryl rings. (An aryl ring is either a simple
phenyl ring or else a phenyl ring with a special group or groups (in

THE CHEMICAL COMPOSITION OF DYES 163

this case -NH2) replacing hydrogen in one or more places.) Dyes
that are related to a leucobase of this kind are called triarylmethane
dyes. Some of them are of great importance in microtechnique.

400

450

500 550

WAVELENGTH, m|x

600

650

FIG. 19. Graph showing the optical density of a layer i cm thick of basic

fuchsine, 0-00062% aqueous.^*

Another colourless derivative of pararosaniline, in which -OH
replaces the hydrogen attached to the central carbon atom, may be
regarded as a triaryl derivative of methanol. Compounds of this

NH, NH,

H

NH2

Leuco-pararosanilinc

kind are called leucohases. This v^ord has unfortunately been
wrongly applied in recent years to Schiff's reagent, which is a
colourless derivative of basic fuchsine but not a leucobase (p. 308).

164 DYEING

Pararosaniline owes its colour to the cation. Dyes that have this
character are called basic dyes. Since the anion is commonly
chloride, they may be shortly represented by the formula R"C1~,
though other anions (sulphate, nitrate, acetate, or oxalate) are
sometimes used instead in basic dyes, without influencing the
colour. Many dyes, however, owe their colour to the anion. Since
sodium is then usually the cation, these acid dyes may be repre-
sented by the short formula Na R". These dyes are often closely
related to basic dyes, but they contain one or more acidic auxo-
chromes, to which the negative charge on the dyeing ion is due.
The auxochrome is commonly the sulphonic group, -SOg".

To convert basic fuchsine (pararosaniline mixed with rosaniline)
to its acidic counterpart, the dye is first treated wdth concentrated
sulphuric acid at about 150° C. Water is then added and the solu-

NH2 NH2

NH2

+
The acidic counterpart of pararosaniline

tion neutralized with calcium hydroxide. The very soluble calcium
sulphonate of the dye is converted to the sodium salt by the
addition of sodium carbonate. Calcium sulphate is precipitated and
may be filtered off. The sodium salt is called acid fuchsine.

It will be noticed that the dyeing ion has a balance of two
negative charges, which are equalized by the two sodium ions.

The transmission of light by acid fuchsine is shown in fig. 20.
Comparison with fig. 17 (p. 161) will show that basic and acid
fuchsine absorb light very similarly. Although their colour is so
similar, their staining reactions are extremely different. (See
pp. 192 to 196.)

The aesthetic desires of man have led to the elaboration of a
huge variety of dyes capable of imparting almost every conceivable
colour to textiles. An impression of the immense number can be
gained by looking through Rowe's invaluable Colour Index,^^^
which gives the chemical composition of each. Structural formulae

THE CHEMICAL COMPOSITION OF DYES 165

for nearly all the dyes used in microtechnique are given in Conn's
Biological Stains, ^^'^ which is produced under the auspices of a
commission set up in the United States to inspect commercial
specimens of dyes used in microtechnique and to certify those that
are suitable for use in particular techniques. It includes a
number of dyes that are not used in the textile industry. The
book gives the wavelength of maximum absorption of many dyes.

100

80 -

52

I 60

z
<

40

20

400 450 500 550 600 650

WAVELENGTH, mp

FIG. 20. Graph showing the transmission of light through a layer i cm
thick of acid fuchsine, 0-00293% aqueous.^*

The use of the full chemical nomenclature for dyes would be
intolerably clumsy, because their structure is usually so compli-
cated. Arbitrarily-chosen names are used instead. Sometimes, as in
the case of light green, the name is a direct statement of the
colour of the dye. The colours of the flowers of familiar plants
give their names to gentian violet, dahlia, and fuchsine. Eosin, as
its name suggests, has a colour reminiscent of the dawn. Sometimes
a certain amount of chemical information is conveyed by a name,
as for instance by naphthol yellow, which is in fact naphthol with
various side-groups. In some cases, however, names that appear
to be partly chemical are in fact misleading. Thus methyl blue
contains no methyl group and methylene blue no methylene

l66 DYEING

group; and azo-carmine is not an azo dye and is not related to
carmine. Some dyes have names of fantastic, almost surrealist
origin. It was discovered that a particular dye was especially con-
venient for the colouring of cotton. In the same year the European
powers recognized the existence of the Congo Free State. These
unconnected events led to the dye being called Congo red.^^^

Despite all that can be said about the lack of consistency in the
naming of dyes, the fact remains that the words used are much
more easily remembered than the numbers and strings of initial
letters that are so often used nowadays in industry for purposes of
this kind, without any thought for those who have to try to hold
in mind which symbol refers to which object.

The names of many dyes are followed by letters or numbers.
These generally serve to distinguish closely-related dyes. Thus
'B' usually means that the dye is more blueish than a related
dye, and 'Y' or 'G' (gelb) that it is yellower. 'WS' conveys that it is
water-soluble, while a related dye is not. The letters A, B, C are
sometimes used as arbitrarily- chosen marks of distinction (for
instance, with the azures, p. 268).

Although dyeing has been and is of such immense service to
biology, the number of dyes that are really useful in microtech-
nique is not very great. There has been a tendency to try new dyes
from mere whim and desire for novelty, and the introduction of
many superfluous ones has been recorded from recipe-book to
recipe-book as though it were the fruit of wisdom. Dabbling with
dyes by persons ignorant of the chemistry of what they are doing
has no counterpart in the rest of science and indeed cannot be
regarded as a scientific activity. The stricture on this subject
reproduced on p. 187 is as applicable today as when it was de-
livered by Gustav Mann more than half a century ago. More so,
perhaps; for it would be a sobering experience to many present-
day dabblers to note the real erudition brought to the subject by
such men as Ehrlich and Paul Mayer, right back in the nineteenth
century. The person who can show a particular dye to be super-
fluous in microtechnique usually deserves better of his colleagues
than he who introduces a new one.

Dyes are classified by their chromophores, but the auxochromes
and modifiers occur over and over again in dyes of diiferent groups.
It is proper to consider these first, for what can be said about them
is of more general application than what can be said about
chromophores.

THE CHEMICAL COMPOSITION OF DYES 167

With a few exceptions that will be mentioned later (p. 262), all

+

dyes are basic or acidic. The fundamental basic group is "NHg.
The usual acidic group is -SOg", and many acid dyes are substi-
tuted sodium sulphonates. A sulphonic acid may be regarded as
sulphuric acid that has lost an oxygen and a hydrogen atom and
replaced them with an organic radicle (R).

Sulphuric acid Sulphonic acid Sulphonate

Other acidic auxochromes are the carboxyl (-Cx ) and

\0H

hydroxyl (-0H) groups, ionized to produce negative charges

(-C\ and -O"). Many dyes possess hydroxyl as well as sul-

phonate groups, and a few (such as fast acid violet A2R) possess

both carboxyl and sulphonate groups. Some dyes have both basic

and acidic auxochromes. The acidic counterpart of pararosaniline

+
(p. 164), for instance, has both -NHg and -SOg", but the negative

charges predominate in number.

In the textile industry it is usual to restrict the name of 'acid
dyes' to those particular acid dyes, in the wide sense, that will dye
wool only from a strongly acid bath, will not dye cotton directly,
and are not used with intermediaries or 'mordants' (p. 207) to
attach them to the fibre. Venkataram, in his invaluable text-
book, ^^^ adopts this nomenclature. It is desirable, however, to
have a single name for all dyes in which the dyeing ion bears a
negative charge, and the term 'acid dyes' will be used in this book
to cover all such dyes.

A variety of different atoms and groups of atoms may replace
hydrogen atoms attached to the aryl and other rings of dyes. Thus
eosin Y contains four bromine atoms, and erythrosine B has
exactly the same composition except that iodine replaces bromine.
These atoms are held by covalent linkages, and there is no question
of the formation of bromide or iodide. The particular atom or
group of atoms attached in this way often affects the colour. The
iodine of erythrosine B shifts the absorption-maximum somewhat
towards the longer wave-lengths, and the dye is therefore bluer
(less red) than eosin.

l68 DYEING

The hydrogens of the amino-groups are often replaced by
methyl, ethyl, and phenyl groups. These particular modifiers tend
to make a red or reddish dye blueish or blue. There are six such
hydrogen atoms in pararosaniline. As more and more of these are
replaced by methyl (in the various dyes that together constitute
the mixtures called methyl and gentian violet), so the colour
becomes bluer and bluer until the hexa-methyl compound, crystal
violet, is reached; this is a very blueish violet dye. Ethyl groups
have more blueing effect than methyl, and phenyl groups still
more, till dyes are reached that are purely blue.

Crystal violet, an important dye for chromosomes, will serve to
show the influence of resonance on colour. When a solution of the
dye is acidified, a proton is added to one of the three dimethyl-

+
HN(CH3)2 N(CH3)2 N(CH3)2

N(CH3)2 2CI- N(CH3)2 Cr

+ +

Acidified crystal violet Malachite green

amino groups, and this now no longer participates in the resonance
of the ion. The resonance is thus limited to that of the related dye,
malachite green; and in correspondence with this, the colour
changes from blue-violet to green.

The more central part of a dye ion also affects colour. If two
benzene rings are put together to form naphthalene, there is more
absorption of electro-magnetic weaves than by two separate
benzene rings, and the same principle applies again when a third
ring is added to form anthracene. Many dyes have a double-ring
component, and a whole group of dyes is based on anthracene

(P- 175)-

CHAPTER 9

The Classification of Dyes

Dyes are classified by their chromophores. In a logical classifica-
tion the natural dyes such as haematein and carmine do not form a
group apart, but divide themselves here and there among the
various groups of dyes, interspersed among the synthetic ones.
Haematein, for instance, owes its colour to a paraquinonoid ring,
and is therefore much more closely related to the fuchsines than
to a dye such as orange G, which owes its colour to an entirely
diiTerent chromophore.

Nearly all the dyes used in microtechnique ow^e their colour
either to a quinonoid ring, or to an azo linkage (-N=N-, p. 182),
or to a nitro-group (-NO 2, p. 184). There are thus three main
groups, of which the first is so diverse that it requires considerable
subdivision, while the third (nitro dyes) is so small that it can be
dealt with very shortly.

THE QUINONOID DYES

These mostly contain a paraquinonoid ring, but in some cases
an orthoquinonoid ring is present instead (p. 181).

II

Paraquinofioid rijig Orthoquinonoid ring

The chief subdivisions of the quinonoid dyes are represented
here by skeleton formulae, from which the auxochromes and
modifiers are omitted. The chromophore is indicated in each case
in a particular resonance-position.

The 'X' in the formula for the azines and related dyes may
represent nitrogen, oxygen, or sulphur. The double bonds of the
quinonoid ring of this group of dyes cannot be represented in full

169

lyo DYEING

in a skeleton- formula, because it is sometimes para- and sometimes
orthoquinonoid.

\

Triarylmethane
/\/0\

Anthraquinonoid
Xanthene

/\/X\/\

I I I I

II Azines and related dyes

Haematein
Skeleton-formulae of the main groups of quinonoid dyes

The following table shows the classificatory positions of the
quinonoid dyes mentioned in this book. The dyes named in heavy
type are the ones that are particularly important in microtechnique.
The table is intended for reference.

TRIARYLMETHANE

Basic. Basic fuchsine (pararosaniline with ros-
aniline), methyl violet, gentian violet,
crystal violet, malachite green, dahlia,
methyl green, aniline blue (spirit
soluble)

Acid. Acid fuchsine, methyl blue, aniline blue
WS, light green

HAEMATEIN

Acid. Haematein

page
171

172

ANTHRAQUINONOID

Acid. Alizarine, alizarine red S, purpurine,
Kernechtrot, carminic acid

175

THE CLASSIFICATION OF DYES 171

page
XANTHENE 178

Basic. Pyronine G

Acid. Eosin Y, erythrosine B, phloxine, fast acid
violet A2R

AZINE AND RELATED DYES (quinone-imine dyes) 179

Oxazine 180

Basic. Brilliant cresyl blue, Nile blue A,
gallamine blue, coelestine blue,

gallocyanine. (The three last-
named are partly acidic.)

Thiazine 180

Basic. Thionine, azure C, azure A, azure
B, methylene blue, new methyl-
ene blue, methylene green, tolu-
idine blue

Azine 181

Basic. Neutral red, safranine O, mauveine,
amethyst violet, Janus green B

(contains also an azo chromophore)
Acid. Azocarmine G, induline (nigrosine)

THE TRIARYLMETHANE DYES

The basic triarylmethane dyes, like basic dyes in general, are
used for colouring chromatin. Crystal violet is one of the best
dyes for chromosomes (p. 225). Methyl green is particularly
valuable in mixtures with the basic xanthene dye, pyronine G,
because it is possible to arrange the proportions in such a way that
DNA is coloured green (or blue), and RNA red (p. 230). Dahlia is
a useful vital dye for various cytoplasmic inclusions.

Basic fuchsine can be converted by sulphurous acid to a colour-
less substance that becomes coloured in the presence of aldehydes.
This forms the basis of certain important histochemical tests
(p. 308).

Acid fuchsine is one of the best dyes for mitochondria (p. 241),
and it can also be used for the differential colouring of collagen,
though other acid dyes of the same group — methyl blue, aniline

172 DYEING

blue WS, and light green — are preferable for this purpose. Acid
fuchsine and light green are also available as background dyes.

HAEMATEIN

This single acid dye has no relative that is commonly used in
microtechnique, but it is so important that rather a full description
is necessary. Indeed, it is probably the most useful of all dyes in
microtechnique. It is used with intermediaries (mordants) between
it and the tissues (p. 207). The importance of haematein derives
from the variety of different objects that can be dyed by it, the
ease with which dyeing can be controlled, the insolubility of the
colour in neutral aqueous and alcoholic media after dyeing, and
the possibility of obtaining a jet black that is permanent in Canada
balsam (such a black being very convenient in photomicrography).

Haematoxylon campechianum Linn, is a small, spreading tree
with crooked branches (fig. 21, a), thorny when young and re-
markable for the gnarled appearance of the stem of old specimens
(fig. 21, c). The stem attains a circumference of about two feet. It
is a leguminous plant, belonging with the tamarind to the group
Caesalpiniaceae, in which the sepals are nearly or quite separate,
instead of being fused as in the pea and its relatives. The pods and
heart-shaped leaflets are shown in fig. 21, B. For a fuller botanical
description see Bentley and Trimen.^^

The sap-wood is white, but the heart- wood red. The latter is

". . . what Campeachy's disputable shore
Copious affords to tinge the thirsty web."

(From Dyers' Fleece, quoted by Bancroft.^'^)

The dyeing property of the heart- wood was known to the natives
of Campeche before the arrival of Europeans. The Spaniards
brought the wood to Europe soon after the discovery of America.
It seems to have been brought to England early in the reign of
Queen Elizabeth. Strangely enough, the dye was thought to fade
and a law prohibiting its use, under severe penalties, was in force
for nearly a century.*^ Its virtues were eventually recognized,
however, and the tree was introduced in 171 5 into Jamaica, where it
is still cultivated at the present day ; for haematein still survives the
severe competition of modern synthetic products in the dyeing of
black on wool, silk, leather, and nylon. Extracts of logw^ood were
first used in microtechnique in the eighteen-forties.^^^

FIG. 21. Haematoxylon campechiamitn.

A, a small, much-branched specimen in the Peradeniya Botanic Garden, Cey-
lon. B, leaves, flowers, and fruit. The side of the square photograph represents 12
inches, c, part of the trunk of a mature specimen in the ^Museum of the Royal
Botanic Gardens, Kew. The white strip of cardboard is 10 cm long.

(a and B are photographs by Mr T. B. Worthington, gth Jan. 1956; c, by the
author, is reproduced by kind permission of the Director of the Gardens.)

THE CLASSIFICATION OF DYES 173

The tree is commonly felled at the age of about ten years. The
bark and sap-wood are chipped off and the heart-wood exported as
'logwood' in pieces three feet long. These logs are reduced to
chips for the extraction of the substance, haematoxylin, from which
the dye originates. Haematoxylin itself is a colourless solid, but if

CH,

O

Haematein

damp becomes oxidized by atmospheric oxygen to the reddish
dye, haematein. It is partial oxidation that gives colour to logwood.
Products in which various proportions of the parent substance
have been oxidized to the dye are available for use in the textile
industry. The biological significance of the presence of haema-
toxylin in the wood is unknown.

The second e in the word haematein (four syllables) dis-
tinguishes this dye from an entirely unrelated coloured substance,
haematin, the non-protein component of haemoglobin. The word
hematine is used in the textile industry for partly or wholly
oxidized haematoxylin."^

Haematoxylin is the leuco-counterpart of haematein. The ring
represented in the formula as quinonoid in haematein is non-
quinonoid in haematoxylin. The oxygen attached to the ring that
is shown as quinonoid in haematein is replaced by -OH in haema-
toxylin. The carbon atom attached by a double bond to the upper
end of this ring in haematein has one valency free in haematoxylin,
and this is satisfied by hydrogen. In all other respects the formulae
for the two substances are the same. Haematoxylin crystallizes
with three molecules of water. ^^^

Many different ways of oxidizing haematoxylin in the laboratory
have been suggested.^^^'"^^^' 2"^' ^-^ Sodium iodate is as convenient
an oxidizer as any. It requires 0-187 g of this to oxidize i g of
haematoxylin crystals fully. ^^^

One might think it best ahvays to buy haematein and make one's
solutions from this, but in fact haematein solutions lose their
strength rather quickly by flocculation of the products of further

174 DYEING

oxidation, and it is therefore generally best to have some haema-
toxylin present in addition to the haematein, to replenish the dye
gradually through oxidation by atmospheric oxygen. Solutions of
haematoxylin are often allowed to 'ripen' gradually. Long ago
Unna ^^^ used to stabilize his solutions after a time by the addition
of sulphur, which was presumably changed in part to hydrogen
sulphide and thus acted as a reducing agent; when he wanted a
strong haematein solution, he fully oxidized a part of the stabilized
solution. A simpler plan is to oxidize a haematoxylin solution only
partly, by the use of less sodium iodate than would be necessary
for full oxidation. ^^ One half or one quarter of the full amount of
the oxidizer is suitable. The solution is ready for use directly it has
been made up, and maintains its strength for a long time by gradual
'ripening' of the part of the haematoxylin that was not oxidized by
iodate. It is desirable to start with wholly unoxidized (white)
haematoxylin.

The products of further oxidation diffuse more slowly into
tissues than haematein, work less well with mordants, and tend to
precipitate.*^^ It is doubtful whether definite stages of further
oxidation can be distinguished, and such terms as Trioxy-
hdmatein ^^^ are not acceptable without more evidence than we
possess.

If one makes a simple aqueous solution of haematein, one has
an acid dye of a dirty reddish colour. It will be guessed from the
chemical formula that this substance is in fact of weakly acidic
nature, like the related substance, catechol. Most acid dyes, as we

HO
HO

Catechol

have seen, are salts, usually of sodium, but in haematoxylin and a
few others (carminic acid and picric acid among them) the metal is
replaced by hydrogen. For clear contrast with other colours one
requires a dye with rather a narrow absorption band, and there are
so many suitable red acid dyes that haematein is never used in this
form.

As we shall see (p. 193), certain acid dyes become basic when
rather strongly acidified, but here again haematein is useless, for a
different reason. At the pH at which it becomes a basic dye, it
is orange, and for reasons that will be mentioned (p. 229), we

THE CLASSIFICATION OF DYES 175

seldom use yellowish basic dyes in microtechnique. Thus haema-
tein is not used alone. In conjunction with mordants, how^ever, it
gives the blue, blue-black, and black colorations that make it pre-
eminent among the dyes used in microtechnique. This subject of
mordants, however, is so complex that a separate chapter must be
devoted to it (p. 207).

THE ANTHRAQUINONOID DYES

The simplest dye in this group is alizarine, the chief coloured
constituent of madder. This plant-product has been used for dye-
ing since ancient times, especially in the production of 'Turkey-
red'. The synthesis of alizarine in the laboratory in 1869 gradually
put an end to the cultivation of the plant and led to the synthesis
of a number of related compounds, some of which are important
in the textile industry. The 1:2: 4-trihydroxy-compound is
purpunnej which occurs also in madder. Alizarine is too insoluble

O

Alizart7je

for convenient use in microtechnique, but purpurine, though only
sparingly soluble in water and alcohol, is used in histochemical
tests for calcium. Kernechtrot or 'calcium red' is an anthra-
quinonoid dye (or mixture of dyes) of unstated composition,
soluble at about 0-25% in water; it can conveniently be used for
the same purpose. '^^^ A soluble sodium sulphonate, alizarine red S,
can be made from alizarine almost exactly as acid fuchsine is made
from basic fuchsine; it is used for staining chromatin, with a
mordant, in Benda's ^^' ^^ method for mitochondria.

Of far more general use in biology than any of these is carminic
acid, a dye of quite special interest for several reasons. It is more
protean than any other colouring agent, for it can be used as a
direct basic dye, is of great value as an acid dye used with a mor-
dant, and is also capable of being taken up directly as an acid dye
and afterwards changed in the tissues to a basic one. The mor-
danted dye is excellent for chromatin, remarkably permanent in
Canada balsam, and particularly suited to the colouring of whole
mounts.

176 DYEING

Carminic acid is the only dye used in microtechnique that is of
animal origin. It is obtained from the scale- insect, Dactylopius
cacti. The female of this animal is wingless and lives on the succu-
lent plant, Nopalea coccinellifera (fig. 22), a native of Central
America. Like other scale-insects, she produces a whitish waxy
material from the surface of her body.^^ The male is winged and
contains only a little of the colouring matter, but the latter is so
abundant in the females that it constitutes about 10% of their
dry weight. ^^^ The Central American Indians cultivated the
animals for the sake of the dye from remote times. The plant on
which it lives is nowadays grown in the Canary Islands, North
Africa, and elsewhere, as food for the insect. The dye has never
been synthesized. It is less used in the textile industry than form-
erly, on account of the competition of synthetic products, but it is
still used for uniforms and hunting 'pink'.*^^

On opening the body-cavity of the fresh female, it can at once
be seen that the colour is in the lobulated fat-body.^^^ A red pig-
ment is contained in globules situated near the periphery of the
cells. These globules give reactions to metals that are characteristic
of carminic acid. The male has as much fat-body as the female, but
there are only a few red globules scattered here and there in it. The
yolk of the ripe egg also contains red droplets, and the colour is
carried over into the embryo and then develops anew in the fat-
body. No other part of the animal contains the dye. The contents of
the alimentary canal are not red. It is clear that the animal syn-
thesizes the substance, but for what purpose is not known (con-
ceivably to neutralize some poisonous constituent of the sap of the
plant, which forms its only food).

The dried females constitute cochineal. To extract carminic
acid from this, the material is powdered and boiled in water, and
the fluid then filtered. On the addition of lead acetate, a dark
red- violet precipitate is formed. This is the lead salt of the dye. It
is dried and ground up with strong ethanol. On the addition of
concentrated sulphuric acid, lead sulphate is formed and carminic
acid dissolves in the alcohol. The yellowish red solution is evapor-
ated at a moderate temperature. (Heat alters carminic acid. It be-
becomes amorphous and eventually insoluble in alcohol.) The
material is purified by repeated extraction with benzene or other
suitable solvents of contaminating substances, and finally allowed
to crystallize slowly from ethanol. *^^ The pure acid consists of red,
prismatic crystals.

A

YOUNG EGGS

FAT BODY

B

WAX
GLANDS

INTESTIN

OVIDUCT

MALPIGHIAN
TUBULE

TRACHEAE

FIG. 22. The cochineal insect and its food-plant.

A, Dactylopius cacti, female and male. B, transverse section through the abdomen of a
female, c, Xopalea coccinellifera, a specimen in the Oxford Botanic Gardens. The white
strip of cardboard is lo cm long. D, female D. cacti attached to A', coccinellifera.

(A from Hartwig; -^' B from Mayer; ^^^ c, photograph by the author, reproduced by
permission of Prof. C. D. Darlington; D from Blanchard.^")

THE CLASSIFICATION OF DYES 177

Carminic acid is a complex substance. It was shown long ago ^^^
that treatment with hot 50% sulphuric acid splits off a side-group
containing six carbon atoms; that subsequent heating to lyo"" C in
a mixture of sulphuric acid with one-third its volume of w^ater
splits off a carboxyl group ; and that a methyl-trioxyanthraquinone

CO(CHOH)4CH3
OH

Carminic acid

is left. The six-carbon side-group is a methyl-pentose sugar, w^hich,
when free, has the formula C^HqO^.CH^.^^^ This is an amorphous,
bitter-sweet, honey-coloured substance. It is claimed that carminic
acid should not be regarded as a glucoside, because the linkage
of the sugar with the anthraquinone is through a carbon atom of the
sugar, not an oxygen atom.^^^

A crude form of carminic acid is often obtained by extracting
cochineal with water and precipitating with alum. The product,
carmine, contains only about 56% of carminic acid, with a high
proportion of protein; also some aluminium and calcium combined
with a part of the carminic acid, and other substances in smaller
amounts. ^^^ Carmine has the advantage of being considerably
cheaper than pure carminic acid. The impurities render it almost
insoluble in distilled water. It is suitable for use in ordinary
microtechnique, but should not be used when one wants to know
exactly what one is doing.

Alcoholic solutions of cochineal were used in the study of plant
stems so early as 1770,^^^ and a few years later Baron von
Gleichen ^^'* was feeding ciliates on carmine, in an investigation
of their method of nutrition. Carmine was first used as a dye in
microtechnique in 1849, by Goeppert and Cohn.^^^ In the
following years its use was repeatedly rediscovered, and the
general recognition of the value of dyeing in biological micro-
technique was due to researches made with this substance. ^^

Pure carminic acid was first used in microtechnique in 1884,^**
but it was Mayer ^^^ who gave it popularity by introducing some
excellent dye-mixtures containing it.

Carminic acid is soluble in both water and ethanol. It is a fairly
strong acid, capable of setting free carbon dioxide from marble.

M

lyS DYEING

It forms blueish red or violet salts with metals. The salts with the
heavy metals are insoluble, but those with the alkali metals are
readily soluble, and indeed carmine itself is soluble in solutions of
borax or ammonia.

The use of carminic acid (or carmine) as a basic dye, and as an
acid dye converted in the tissues to a basic one, is described on
p. 193. For its use with mordants, see chapter 11 (p. 207).

THE XANTHENE DYES

These are to be regarded as derivatives of xanthene, which
itself exists in the form of colourless leaflets. A few dyes related to
this substance occur in nature, including a yellow one extracted in

/\/0\/\

I I I I

Xanthene

India from the urine of cows that have fed on mango leaves ; but
the xanthene dyes in the strict sense are synthetic products. A para-
quinonoid ring gives colour; an oxygen atom participates in the

+
/\ O ^\^N(CH3)2 Cl-

H

Pyronine G

linking of two rings. The simplest of those that are useful in micro-
technique is the basic dye pyronine G, which, as has already been
mentioned (p. 171), is used with methyl green in histochemical
tests designed to distinguish DNA from RNA.

A far more familiar dye in the laboratory, and indeed one of the
best known of all that are used in microtechnique, is eosin Y. This
is unusual among synthetic acid dyes in not being a sulphonate.

The auxochromes, as the formula shows, are -Q and -O . It

is used chiefly as a background dye. Its yellowish red colour and
small capacity to overstain fit it rather well for this function, especi-
ally when blue dyes such as aluminium-haematein (p. 215) are
used for chromatin. A special advantage of eosin is that the dye is

THE CLASSIFICATION OF DYES 179

able to penetrate red blood-corpuscles and other close-textured
components of tissues. This is so despite the large size of the dyeing
ion. The capacity to penetrate well must be ascribed to the tend-

Br Br ^

Til Jx J\^Br

^O- Na+

Eosin Y

ency of the ions to remain separate instead of aggregating (p. 236).
Eosin Y can be made to stain red blood-corpuscles most power-
fully, and show them up so vividly that the course of the capillaries
can sometimes be traced as easily as in an injected specimen.
Mann's methyl blue/eosin (the long method) is particularly well
adapted to this purpose. ^^^

Phloxine and erythrosine B are background dyes closely re-
sembling eosin Y. The former has chlorine atoms as well as
bromine; er}'throsine B is exactly the same as eosin Y except that
four iodine atoms are substituted for the four bromines. In both
of these, especially phloxine, the greatest absorption of light is
shifted towards the longer wavelength, with a resulting bluer (less
yellow) shade.

AZINE AND RELATED DYES

(quinone-imines) '

These dyes include some of the most valuable in microtechnique.
The chromophore is a quinonoid ring associated with a substi-
tuted imino-group (HN=). They may be regarded as derived from
quinone-imine by the substitution of an aryl ring for the H of the
imino-group, and they are therefore sometimes called quinone-
imine dyes. In the simplest of these the two rings are connected

I i ^^11

Quinone-imine Indophenol

by a single link only (-N=). The indophenol dyes, which are
briefly mentioned on p. 288, are of this nature. In all the quinone-
imine dyes that are important in microtechnique, however, there

l8o DYEING

is a second link between the two rings, through an atom that may
be oxygen, sulphur, or nitrogen. The three groups of dyes are
respectively the oxazines, thiazines, and azines.

Oxazines

The link through oxygen will be noted in the structural formula
for the basic vital dye, brilliant cresyl blue. Among the small
group of oxazine dyes used in microtechnique, a high proportion

+

pj pi I I I

Brilliant cresyl blue

can be used vitally. This applies to Nile blue A, which is also used
in a roundabout way in histochemistry for the distinction between
certain groups of lipids (p. 301).

Several of the oxazine dyes, coelestine blue B among them, can
be used w^ith a mordant (p. 215).

Thiazines

In these dyes, several of w^hich are among the most important
used in microtechnique, the second part of the link between the

H n/\/S\^\^NH, CI-

I I I I

Thio7iine

two rings is formed by an atom of sulphur. The simplest is thi-
onine, a violet dye. The thiazine dyes used in microtechnique are
all basic.

The four hydrogens attached to nitrogen can be replaced by
methyl groups. One is so replaced in azure C, two in azure A,
three in azure B, all four in methylene blue (see p. 268). As the
replacement occurs, so the absorption maximum shifts towards the
longer wave-lengths, and methylene blue is a pure blue dye.
Toluidine blue is closely related to azure A.

The thiazine dyes are useful for staining chromatin. Their chief
virtue, however, is that they have a particularly strong tendency
towards metachromasy: that is to say, towards the dyeing of
different tissue-constituents in different colours. This subject is

THE CLASSIFICATION OF DYES l8l

discussed elsewhere (p. 243). It is their metachromatic property
that makes these dyes so important in the staining of blood-
corpuscles (p. 268).

Methylene blue is a valuable vital dye (p. 287).

Azines

The structural formulae for all the diverse dyes that have been
mentioned so far can be written down so as to include a para-
quinonoid ring, and this has been done. In some dyes, however,
there may in fact be resonance between the paraquinonoid and
orthoquinonoid configurations, and for the azine dyes it becomes

1+

Skeleton-formula for azines

necessary to write orthoquinonoid formulae. In this group an
imino-nitrogen once more forms one of the links between two
rings, but, in contrast to the oxazines and thiazines, the second
link is also formed by a nitrogen atom. The skeleton-formula shown
here is satisfactory for most of the azine dyes. Most of the ones

H,cl ' ^1-

Safranine

used in microtechnique are rather complex. The safranines have
an aryl ring attached to the charged nitrogen atom. Safranine O, a
useful red dye for chromatin, consists of the dye shown here mixed
with another dye diflfering only in the absence of the methyl
group from the attached aryl ring.

Two other basic azines, neutral red and Janus green B, are of
outstanding importance as vital dyes. The former, a dull red, is an
exceptionally innocuous dye that colours certain kinds of globules
in the cytoplasm. It turns yellow on the alkaline side of neutrality,
and one can thus learn something of the acidity or alkalinity of
cytoplasmic inclusions by using it. Janus green B, a very complex
dye that contains also an azo chromophore, has been much used
in the study of mitochondria (p. 292).

l82 DYEING

A few acid dyes of this group are important for the production of
na\y blue on wool. Only two or three are used in microtechnique.
Azocarmine G, red, is exceptional among acid dyes in being used
for colouring chromatin (p. 205). Induline WS, blue-black, is
useful for collagen. This is an acid dye related to safranine, but
with the hydrogens of the amino-groups replaced by phenyl
rings. Usable specimens, blended with other dyes to produce a
blacker colour, are sometimes sold as nigrosine, and this name
occurs frequently in the older literature of microtechnique. There
is unfortunately considerable variation among dyes sold as nigro-
sine, and some of them are unsatisfactory as collagen-dyes.

THE AZO DYES

We now leave quinonoid rings behind us and pass to an entirely
different chromophore, -N=N-. The azo dyes nowadays occupy
a predominant place in the textile industry. A huge variety of reds,
oranges, and yellows is available, as well as other colours. The
number of acid azo dyes suitable for the direct colouring of wool

Skeleton formula for azo dyes

exceeds that of all such acid dyes of other groups put together.
Many azo dyes can be used with mordants, and about three-
quarters of all the dyes that are used with mordants in the textile
industry are of the azo-group. The number of azo-dyes that are
useful in microtechnique is, however, rather limited.

It is a remarkable fact that no living organism produces an azo
dye. They are made synthetically by the action of nitrous acid or
nitrites on amino-derivatives of benzene. If aniline hydrochloride
is treated with sodium nitrite, a nitrogen atom leaves the sodium

13 ^1 -r \ yl

">— NH3 CI- + < >NH3 Cr + NaNOs

Aniline hydrochloride Aniline hydrochloride Sodium nitrite

— N=N— <^ ^NHg CI- + NaCl + 2H2O
Amino azohetizene hydrochloride

nitrite and associates through a double bond with a nitrogen atom
of an amino-group, thus introducing itself as a link between two
rings. The resulting ion gives a yellow basic azo dye.

THE CLASSIFICATION OF DYES 183

Dyes that contain a single -N=N- group are called mono-azo;
two, disazo; and three, trisazo. The azo dyes mentioned in this
book are listed below, for ease of reference. The ones that are
particularly important in microtechnique are named in hea\y
type.

MONO-AZO

Basic. Janus green B (contains also an azine chromophore)
Acid. Orange G, chromotrope 2R, ponceau 2R (= xylidine

red), Bordeaux red, metachrome yellow RA, trop-

aeolin, methyl orange

DISAZO

Basic. Bismarck brown Y

Acid. Congo red, Congo rubin, trypan blue

TRISAZO

Acid. Chlorazol black E

MONO-AZO DYES

Several acid mono-azo dyes, of the yellow, orange, or red colour
that is so usual among azo compounds, are useful for background
coloration. Examples are orange G, chromotrope 2R, and ponceau
2R (= xylidine red). These three dyes are closely related. Orange

-o.,s<^

-N=N— <^
Orange G

G is of a colour near the middle of the visible spectrum that con-
trasts well with the familiar blue dyes for chromatin, and has the
further advantage that it is very unobtrusive: that is to say, it has
little tendency to overstain or to apply itself to the objects that are
coloured by basic dyes.

Metachrome yellow RA is a simple azo dye, remarkable in more
than one respect. It is an acid dye that has carboxyl and hydroxy!
groups for its auxochromes, and also possesses a nitro-group;
it is scarcely soluble in water, and therefore lends itself, unlike
other acid dyes, to the background staining of specimens that will
be mounted in aqueous media.

184 DYEING

The only important basic mono-azo dye that is important in
microtechnique is Janus green B, which, since it contains an azine
as well as an azo chromophore, has already been mentioned (p. 181).

DISAZO DYES

Bismarck brown has the distinction of being the first synthetic
vital dye that was ever used (p. 274). The colour is distinctly
unusual in microtechnique. For most purposes it is desirable to
use dyes that have either a rather sharply marked absorption-band,
so that strong contrasts with other colours can be arranged, or
else a general absorption throughout the visible spectrum to give
a black that will contrast with an unstained or lightly stained back-
ground. Thus browns and yellowish browns are seldom chosen.
Bismarck brown Y is a basic dye, convenient for colouring lipid
cytoplasmic inclusions during life.

Trypan blue and its relatives are acid dyes that are used in a
very special way in a particular kind of vital work (p. 276).

Several coloured disazo compounds that are not dyes, because
they do not ionize, are very useful in microtechnique for colouring
lipids (p. 299).

TRISAZO DYES

The only trisazo dye that has found favour in microtechnique is
chlorazol black. ^^- It is an acid dye with some basic tendency.

THE NITRO DYES

The third and last main chromophore with which we shall be
concerned in this book is the nitro-group, -XOg. Colour is due to
resonance between two possible positions of the negative electric
charge. Nitrobenzene, like other substances containing -NO 2, is

Nitrobenzene in two resonance positions

coloured. It is a yellow liquid, but lacks an auxochrome and so is
not a dye. Trinitrobenzene possesses three chromophores but is
not a dye. If phenol be mixed with concentrated nitric acid, how-
ever, a yellow crystalline substance is formed, which, because it
contains both -NO 2 (three times) and the auxochrome -OH, is a

THE CLASSIFICATION OF DYES 185

dye. It is trinitrophenol or picric acid, the only substance that is
used both as a fixative (p. 96) and a dye. It is one of those acid
dyes that are actually acids, like carminic acid, for instance. Its

OH

OaNj^NNOa OaN^NNOa

NO2 NO2

Trinitrobenzene Picric acid

salts are seldom used in microtechnique. It is a far stronger acid
than phenol, and this to some extent militates against its use as a
background dye, for which its pale yellow colour otherwise fits it ;
for it has a tendency to remove basic dyes. Its acidity, however, is
not harmful to the action of other acid dyes, and it is useful in
such mixtures as picro-nigrosine (p. 236).

The only other nitro-dyes that are at all frequently used in
microtechnique are naphthol yellow and aurantia. The latter is
the background dye in Kull's ^"^ method for mitochondria.

Orcein is a dye of considerable interest. It has long been used for
showing elastin (p. 233) and latterly has come into favour in tech-
niques for showing chromosomes in smeared preparations. It
cannot be included in the classification of dyes, because its struc-
tural formula is unknown.

Since ancient times dyes have been prepared from lichens. The
modern usage, however, dates from the fourteenth century, when
a Florentine merchant began to make them on a large scale.
Rocella, the genus most commonly used in the preparation of
orcein, commemorates the name of his family. The lichens of this
genus, commonly called orchil- or archil-weed, grow chiefly on
rocks near the sea-shore in the warmer parts of the world. They
form tufts of bluish-grey or whitish strap-shaped fronds, up to
6 inches long. Several species are usable as raw materials for the
preparation of orcein. In Scandinavia and other cool climates
another lichen, Lecanora tartarea, replaces Rocella^ and indeed
many other species can be used.*^^ Lecanora is often called cud-
bear, but the familiar names are loosely used.

These various lichens contain lecanoric acid, which splits to
produce orcinol when the plants are boiled with water. ^^^ It will
be noticed that orcinol is resorcinol with a methyl group attached.
It crystallizes as colourless, hexagonal prisms, freely soluble in
water. The substance can also be prepared synthetically. ^^^

l86 DYEING

In the presence of ammonia and atmospheric oxygen, orcinol
becomes transformed into orcein, which precipitates. Stale urine
was formerly used as a source of ammonia. Orcein is sold as a fawn-
coloured powder, soluble in alkaline or alcoholic solution or in

HO^c/\ HOAOH UOfyu ^

CH

CH3

o

Lecanoric acid Orcinol

acetic acid. It is used for dyeing wool and silk directly (that is,
without the use of mordants). It gives dull magenta shades, but is
often used with other dyes to give browns. ^'^^

The formula for orcein is probably C28H24N2O7. It has been
suggested that the two nitrogen atoms may form the chromophore
of an azine dye.^^^

Litmus is produced from the same lichens as orcein by a closely
similar process, but potassium carbonate is required in addition to
atmospheric oxygen and ammonia.^^^' *^^

The indigo-dyes are briefly mentioned on p. 307, the acridine
dyes on p. 310.

i

CHAPTER 10

The Direct Attachment of Dyes

to Tissues

An insoluble pigment requires the addition of an adhesive if it
is to remain in position when applied to any object. A dye is pre-
sented in solution without the addition of any adhesive ; it is not
itself sticky or obviously adhesive; yet it adheres. It is the purpose
of this chapter to explain the reason for this.

It has already been mentioned (p. 172) that certain dyes are
used with intermediaries between themselves and the tissues.
These intermediaries or mordants are not adhesive in any
ordinary sense. Chapter 11 (p. 207) deals with the use of these
substances.

The nature of the process of dyeing has been studied more
elaborately by the research-workers of the textile industry than by
those who use dyes in microtechnique. This is due partly to the
fact that larger funds are available in industry and partly to the
relative homogeneity of the textile fibres — especially cotton — in
comparison with the kinds of tissues usually studied by biologists.
It must be confessed also that the practical dyer has generally
adopted a more scientific approach to his work than the histologist
and cytologist. As Mann ^^^ unkindly remarked: —

'The method of staining, once having taken root in the animal
histologist, grew and grew, till to be an histologist became
practically synonymous with being a dyer, with this difference,
that the professional dyer knew what he was about, while the
histologist with few exceptions did not know, nor does he to the
present day.'

The realization of this truth is helpful to the biologist, but it is
necessary to point out another truth that has been overlooked,
namely, that there are many very important differences between
the dyeing of textile fibres and the dyeing of tissues in biological

187

i88

DYEING

microtechnique. This is true although some of the most important
textile fibres are those of plant and animal origin. The chief
differences are tabulated here.

Textile dyeing

Cotton has been especially in-
vestigated, because it is so homo-
geneous chemically. It is extreme-
ly peculiar, because it is a nega-
tively charged object ordinarily
dyed by acid dyes.

Whether, in particular circum-
stances, a dye is acting as a basic or
an acid dye may not be known
(p. 209).

Dyes are generally used at or
near the boiling point of water.

The dye-bath is usually ex-
hausted or nearly so.

Almost perfect fastness to water
is generally required.

The fibres — of cellulose or spe-
cial proteins, or synthetic — are
non-living and unfixed.

No differential dyeing at the
microscopical level is required, and
there is no process of differential
extraction of the dye.

Anionic chromium is used to
mordant for azo-dyes; other mor-
dants and mordant-dyes are seldom
used nowadavs.

Dyeing in microtechnique

The dyeing of cotton is of little
interest except to those studying
the cell-walls of plants. The bio-
logist does not ordinarily dye
negatively charged objects with
acid dyes.

A glance down the microscope
at a dyed preparation usually
shows whether the dye used was
basic or acid.

Dyes are usually used at room
temperature.

The tissue takes up only a
minute part of the dye in the dye-
bath.

Fastness to water is unneces-
sary, as one can quickly transfer
the tissue to some other medium
in which the dye is insoluble.

The objects dyed are generally
either fixed or alive (p. 274).

The whole purpose is differ-
ential dyeing at the microscopical
level; it is often achieved by
differential extraction.

Iron, aluminium, and cationic
chromium are used to mordant
for haematein, carmine, and cer-
tain oxazine dyes (see p. 207).

In this book every effort will be made to profit from the valuable
researches of the textile chemists, and it is necessary to make
special acknowledgement of the admirable presentations of this
subject by Vickerstaff,^^^ Bird,^^ and Venkataram; ^-^ but the
object throughout wall be to concentrate attention on the use of
dyes in microtechnique.

In microtechnique we are primarily concerned wdth the electric
charges on the dye-ions and on the objects dyed.

The electric charges on dye-ions are investigated by subjecting
dye-solutions to the action of an electric current. Cataphoretic

FIG. 23. Apparatus for cataphoretic experiments with dyes.^** The wires

from a 12-volt accumulator are marked — and +. Methylene green (a

basic dye) is being tested. The current has been passing for 42 hours. For

full description see accompanying text and Appendix (p. 321).

(Photograph by Mr P. L. Small)

THE DIRECT ATTACHMENT OF DYES TO TISSUES 189

experiments of this sort have been carried out especially by
Seki,*^2 a Japanese investigator who has played a particularly
important role in the scientific study of microtechnical dyeing.
An apparatus resembling Seki's is shown in fig. 23; practical
instructions for setting it up are given in the Appendix (p. 321).

The main part of the apparatus is the large U-tube in the middle
of the photograph. This contains an aqueous agar gel. It fills the
tube up to the level marked 'O' on the cardboard scale fixed beside
the tube: the level is also marked by a line on a label stuck on to
each limb of the tube. The gel is bufi"ered at a known pH. The dye-
solution, buffered at the same pH, is poured into both limbs of the
U-tube to the same height. In principle, one might now put the
positive wire from an accumulator into the dye solution in one
limb and the negative wire into the dye solution in the other, and
the experiment would begin. If this were done, however, the
electrolysis of water would take place, gas would be formed at the
electrodes, and the latter would become depolarized. The rest of
the apparatus exists solely to prevent this. It consists of two beakers
into which dip electric wires (marked -f and — ), and two small
U-tubes, each of which dips on one side into a beaker and on the
other into the dye in the big U-tube. The current passes through
agar gel in the small U-tubes. The contents of the beakers and
U-tubes are given in the Appendix.

The electric current should be switched on as soon as the dye
has been poured into both sides of the big U-tube. The current
now tends to make the dye move towards either the negative or the
positive pole: that is to say, to make it descend in one or the other
of the two limbs of the U-tube. Simple diff"usion also occurs, how-
ever, and this causes some descent on both sides. When basic dyes
are used, there is also an attraction between the dye and the agar,
which causes some descent on both sides. The much greater
descent on one side than on the other shows that the dye moves in
response to the current. In the figure the dye in question (methyl-
ene green) has moved in 42 hours about 6 cm towards the negative
pole and only about 2 cm towards the positive. The dye ions are
clearly positively charged or cationic, and indeed methylene green
is a cationic or basic dye.

This apparatus enables us to find whether any dye is basic or
acid at any particular pH. In general, any dye that is shown by its
chemical formula to be basic will behave like methylene green,
while any acid dye will move in the opposite direction. The speed

190 DYEING

with which the various dyes move varies considerably. Thus
methylene green is one of the fastest, dahlia one of the slowest of
the basic dyes."*^^ This depends partly on the electric charge,
partly on diffusibility.

Most dyes remain cationic or anionic, as the case may be,
throughout the range of acidity and alkalinity within which dyeing
ordinarily takes place in microtechnique: that is from about pH3
to 9. Examples of basic dyes that are typical in this respect are
crystal violet and safranine, while orange G and picric acid are
typical acid dyes.*^^ Some dyes, however, are amphoteric, being
cationic below a certain pH (the iso-electric point) and anionic
above. Examples are lithium carminate and haematein, with iso-
electric points about pH 4-5 and 6-6 respectively.^^^ These facts
facts can be expressed in a simple diagram.

pH3 4 5 6 7 8 9

Typical basic dye. + + + + + + + + + + + + + + + +

An amphoteric dye. + + + ________ — __

Typical acid dye • ________________

The majority of dyes fall within this scheme, though some are
bleached by acidity and more by alkalinity, so that their behaviour
cannot be studied cataphoretically at the ends of the pH range.

Electrically-charged groups occur also in the tissues, especially
in the proteins and certain lipids. The most obvious sources of

electric charges in the proteins are the -C<^ and -NHg

groups of certain amino-acid residues. A part of a protein chain at
the iso-electric point is here represented by an example.

HC(CH2)2C\ glutamic acid

?

NH

HC(CHo)4NH2 lysine

Part of a protein chaitt at the iso-electric point

THE DIRECT ATTACHMENT OF DYES TO TISSUES I9I

The degree of ionization of the carboxyl and amino-groups
depends on the pH. When this is pushed to the acid side of the
iso-electric point, the dissociation of the acid groups is suppressed
and that of the amino-groups increased. The protein thus becomes
progressively more positively charged as more and more of the
amino-groups become ionized. They are all ionized when pH 2 is
reached, or thereabouts.

NH

HC(CH2).C(

NH

I +

HC(CH2)4NH3 ionized amino-group

^

=0

Part of a protein chain on the acid side of the iso-electric point

When the pH is on the less acid or more alkaline side of the
iso-electric point, the dissociation of the amino-groups is sup-
pressed, while that of the carboxyl groups is increased. Thus the
protein becomes more negatively charged as the pH increases,
until all the carboxyl groups are ionized at about pH 11.

NH

I ^O . .

HC(CH,)2Cx ionized carboxxl group

T " ^o-

o

?

NH
HC(CH2)4NH2

T

Part of a protein chain on the less acid or more alkaline side of the iso-electric

point

Although the protein as a whole is neutral at the iso-electric
point, yet a few ionized carboxyl and amino-groups still exist at
this pH. This is important for dyeing.

The chief amino-acid side-groups that can give negative charges
are aspartic, glutamic, and hydroxy-glutamic. The -OH group of
tyrosine and serine can also be ionized, and so can the terminal
carboxyl group of a protein chain.

192 DYEING

The amino-acid side-groups that can give positive charges are
lysine, arginine, and histidine, and the terminal amino-group of
the chain is also available.

The majority of the amino-acids (glycine, alanine, leucine,
phenylalanine, etc.) cannot provide electric charges unless they
happen to lie at the end of a chain.

Since different proteins contain different proportions of acidic
and basic amino-acids, the position of the iso-electric point
varies.

The tissues provide a number of other charged groups beyond
the amino-acid residues of proteins. Negative charges can occur on
the phosphoric acid groups of nucleic acids and phospholipids, and
on the uronic and sulphuric groups of mucopolysaccharides.

The various tissue-constituents that are visible under the micro-
scope vary in their content of these positively and negatively
charged groups. Some are preponderatingly positive or basic,
others negative or acidic, others again amphoteric or easily swayed
by changes in pH. Some characteristic examples are these: —

Acidic . . DNA and chromatin

RNA and ribonucleoprotein

matrix of cartilage

many mucous secretions

most Hpids other than triglycerides

Amphoteric . cytoplasm of most cells

contractile substance of muscle

Basic . . collagen

cytoplasm of red blood-corpuscles

granules of eosinophil leucocytes

nuclei of the spermatozoa of certain fishes.

The substances here listed as acidic and basic act as such within
the range of pH at which dyeing usually takes place in micro-
technique, but if the dye-solution be made sufficiently acid or
alkaline it will be found that they are in fact amphoteric.

Since both dyes and tissue-constituents are electrically charged,
it is natural that they react with one another. The acidic constitu-
ents have an affinity for basic dye-ions and are therefore called
hasiphil (see p. 329), while the basic attract acid dye-ions and are
called acidophil. The fact that basic dyes show a 'most striking con-
formity with one another' in their reactions with tissue-constitu-

FIG. 24. Ehrlich at the age of 24. At about this age he dis-
covered the fundamental differences between basic and acid
dyes in their reactions with the constituent parts of cells, and
also showed how the varying permeability of these parts
aided differential dyeing,

(From Marquardt,^-* by kind permission of Messrs William Heine-
mann Medical Books, Ltd.)

THE DIRECT ATTACHMENT OF DYES TO TISSUES I93

ents was remarked by Ehrlich ^^'^ in 1879. ^^ ^^^ same year he
noted ^^^ the affinity of acid dyes in general for the granules of
what he had named 'eosinophil' leucocytes. In the following year
he gave a short general statement of the differences between basic
and acid dyes in their reactions with cell-constituents, illustrating
his remarks by reference to leucocytes. ^^* He noted that the gran-
ules of eosinophils have an affinity for acid dyes, of Mastzellen for
basic dyes, and of polymorphs for both constituent parts of
'neutral' dyes (p. 262). These papers, published when Ehrlich
was about 25 years old, mark a turning-point in the history of
scientific microtechnique.

In preliminary studies of the reactions of dyes with electrically-
charged objects it is convenient to use simple, homogeneous
models to represent the tissue-constituents. The acidic and ampho-
teric constituents are the most worthy of attention, because every
cell contains them. Seki *^^ chose collodion as a model for the
acidic components, gelatine gel for the amphoteric. These sub-
stances are very convenient, because they can so easily be cut in
slices of uniform thickness, but collodion is more acidic than
most tissue-constituents. The depth of colour may be recorded on
an arbitrary scale, or determined by photometry. Practical instruc-
tions for some experiments resembling Seki's are given in the
Appendix (p. 323).

The results with collodion (fig. 25) are simpler than those with
gelatine because the former, when in water or aqueous solutions,
maintains its negative charge throughout the relevant range of pH.
It is strongly dyed by crystal violet or any other typical basic dye
throughout the range. Typical acid dyes, however, scarcely tinge
collodion, except in strongly acid solutions, in which the charge on
the collodion is somewhat lessened.

An amphoteric dye behaves as one would expect on theoretical
grounds. On the acid side of its iso-electric point it is positively
charged and therefore dyes collodion strongly, but on the less acid
side it becomes negatively charged and its tendency to colour
collodion disappears about neutrality. These facts are well exempli-
fied in the practical use of carminic acid, the iso-electric point
of which is about pH 4-2. It is commonly used for dyeing chro-
matin in the form of aceto-carmine.*'*^ In this strongly acid solu-
tion it acts as a basic dye.*^*' *^^ In alkaline solution, however, in
the form of borax-carmine, ^^^ it acts as an acid dye, but is usually
converted subsequently into a basic one by treatment with acid.*^*

N

194 DYEING

Orcein is another amphoteric dye,*^^ much used in its basic state —
that is, in strongly acid solution — for the dyeing of chromosomes. ^^^
Haematein is yet another amphoteric dye,*^^ but it is not used as
a direct dye in either its basic or acid character, as the colours it
produces are feeble and indefinite. The way in which this im-
portant dye is used in practice will be explained in chapter ii
(p. 207).

BASIC DYE

O
z

o

u- 2

o

>■

in

UJ

PH

FIG. 25. Diagrammatic representation of the dyeing of collodion by
typical basic, amphoteric, and acid dyes. The ordinate is divided
into arbitrary units. In general conformity with the data of Seki.*^^

It was mentioned above that most tissue-constituents are shown
to be amphoteric if tested towards the extremes of pH, and their
reactions to dyes are therefore more complicated than those of
collodion, which remains negatively charged even in strongly acid
solution. More than half a century ago Bethe ^^ tried the efi'ects of
adding varying amounts of sodium hydroxide or sulphuric acid to
solutions of toluidine blue. He tried these solutions on various
mammalian tissues fixed in alcohol. Whereas everything was
colourable in alkaline solutions or at neutrality, there was a tend-
ency for the various constituents to fail to take the dye as more and
more acid solutions were used: one constituent after another dyed
more feebly or failed to dye at all. Cytoplasm fell oflP rapidly in
capacity to be coloured, chromatin much less rapidly and mucus
less rapidly still, while the ground substance of cartilage remained
as deeply dyed in the most acid solution tried as at neutrality.

THE DIRECT ATTACHMENT OF DYES TO TISSUES I95

Pischinger ^^-^ developed Bethe's study by using both basic and
acid dyes, instead of basic only. He showed that the effect of both
basic and acid dyes tended to fall off sharply towards a particular
pH, which he regarded as the iso-electric point of the object dyed.
Thus an acid dye would colour a particular object in the tissues in
strongly acid solutions, but would scarcely act above a certain
pH. A basic dye would act strongly on the same object in alkaline
solution, but the affinity between dye and object would fall and

FIG. 26. Diagrammatic representation of the dyeing of gelatine by
typical basic, amphoteric, and acid dyes. The ordinate is divided
into arbitrary units. In general conformity with the data of Seki.^^*

I.E. P., iso-electric point.

nearly disappear at about the same pH as that at which the acid dye
failed to act. The position of the iso-electric points of tissue-
constituents may indeed be roughly estimated by Pischinger's
method, but different pairs of dyes do not give exactly the same
results, and Lison ^'^^ prefers to speak of the 'apparent iso-electric
point' when referring to information gained in this way.

Instead of using a basic and an acid dye separately, one may mix
them together and judge the position of the iso-electric point of
any particular object in a microscopical preparation by noting the
pH at which the mixed colour is given. ^^^ Methylene blue and
eosin make a good pair for this purpose.

Methods of this sort help in the histochemical identification of
certain tissue-constituents. Carboxyl groups soon lose their nega-
tive charge as the pH is lowered, then phosphoric groups, and the

196 DYEING

sulphuric groups of certain mucosubstances later still. These facts
provide the explanation of Bethe's results.

Seki ^^^ chose sheet gelatine, untreated by any fixative, as a
model for amphoteric tissue-constituents. The dyeing of gelatine
is represented diagrammatically in fig. 26. The iso-electric point of
this substance is about pH 4-7. Since gelatine is positively charged
at lower pH than this, it has a strong affinity for ordinary acid
dyes ('levelling' dyes, see p. 235) in strongly acid solution. In the
same way wool has a strong affinity for these dyes at low pH. The
affinity of gelatine or wool for ordinary acid dyes falls off rapidly as
the pH rises above the iso-electric point, but now% as the charge has
become negative, there is increasing affinity for typical basic dyes.

The curves representing the uptake of acid and basic dyes by
proteins cross somewhere, and the place of crossing is at or near
the iso-electric point of the protein. It might be thought that dye-
ing would be impossible beyond the iso-electric point, so that the
curves for acid and basic dyes w^ould reach the base-line here,
instead of crossing. It must be remembered, however, that pro-
teins are not without electric charges at the iso-electric point: such
charges still exist, but the positive and negative ones balance one
another.

Accurate figures for the amounts of a basic and an acid dye
taken up by thin fibrin films have been obtained photometrically
by Singer and Morrison. ^"^ Their results confirm the general
correctness of the data provided by Seki, who used an arbitrary
scale (+, +-f, etc.) to represent the intensity of dyeing as judged
visually.

The facts just recorded explain the common custom of using
basic dyes in w^eakly acid solution. The pH is low enough to pre-
vent the amphoteric cytoplasm from taking the dye, but not low
enough to prevent the chromatin from being coloured strongly.

The complication is considerable when an amphoteric substance
is treated wath an amphoteric dye. If the iso-electric points of both
were the same, there would be little affinity between them. If,
however, the iso-electric points are different, there will be a
narrow range in which the charge on the dye will be opposite to
that on the protein; dyeing will occur within and to some extent
also beyond this range (fig. 26).

After basic dyes have acted, there is generally little tendency to
extraction by water at neutrality. Many acid dyes are less fast to

THE DIRECT ATTACHMENT OF DYES TO TISSUES I97

water. (IVIetachrome yellow, an acid dye, is exceptional in being
nearly insoluble in water, so that it could not easily escape even if
the bond with the tissue were loosened.) Acids and alkalis release
respectively basic and acid dyes, and once again the iso-electric
point of a particular tissue-constituent will determine whether
or how quickly it will release a dye. These facts form the basis of
regressive dyeing. Instead of allowing the dye to colour the tissues
progressively until the desired effect is obtained, one may over-
stain and then extract the excess of dye differentially from the
various tissue-constituents. The hydronium and hydroxide ions
diffuse through the tissue much more rapidly than any dye-ion
and therefore give more even results, especially if the piece of
tissue be thick.

Although the basic dyes show more resistance to washing out
by distilled water than acid ones, many of them are very quickly
removed by the alcohols used in dehydration, which remove the
acid dyes more slowly. Indeed, the dehydrating alcohols act like
very weak acids. *^^ Their powders of extracting basic dyes diminish
in the series methanol, ethanol, propanol, butanol, pentanol.^^®
There appears not to have been any full investigation of the various
isomers of the three last-mentioned alcohols, but tertiarv butanol
is said not to remove any toluidine blue that has combined with
nucleic acids. ^^" It might be thought that basic substances, such as
aniline and pyridine, would help to hold basic dyes in the tissue,
but in fact they appear to compete with the dye for the acidic
components of the tissues. '^^^

A simple experiment, described in the Appendix (p. 324),
shows that acidified alcohol rapidly removes all colouring of
tissues by a basic dye, but leaves intact the colouring by an acid
dye.

Mollendorff ^^^ considered that a sharp distinction was to be
drawn between the action of acid dyes and certain basic ones on
one hand, and of other basic dyes on the other. The latter, in his
view, were especially liable to flocculation and tended to be
precipitated on the surface of the objects for which they had an
affinity. This process he called precipitation-dyeing [Nieder-
schlagsfdrbiing). Other dyes, not subject to flocculation, penetrated
into the interstices of objects and dyed them uniformly throughout.
He called this permeation- dyeing (Durchtrdnkungsfdrbung). This
distinction is perhaps valid. One sometimes has the impression
that certain basic dyes and dye-lakes (p. 207) have a tendency to

198 DYEING

thicken the objects for which they have an affinity, which would be
impossible if they only acted by permeation.

It is also easy to gain the impression that basic dyes show a
considerable degree of specificity, while acid ones act diffusely.
This apparent contrast is not quite genuine. The difference lies in
the objects dyed rather than in the dyes. If every cell contained a
characteristic object as basic as chromatin is acidic, we should not
be so much struck by the diffuseness of acid dyes, for we could
arrange to colour the basic object differentially with them. The
eosinophil granules of certain leucocytes can indeed be dyed
sharply in this way. Often, however, we deliberately use the acid
dyes to colour the amphoteric cytoplasm feebly, in a colour
contrasting with that of the acidic objects, which have been
strongly coloured by a basic dye.

Despite this, there is reason to believe that acid dyes really are
somewhat more diffuse in their action on tissues than basic ones,
and it is reasonable to look for a component of the protein chain
with which they might react, other than the specifically basic
residues of lysine, arginine, and histidine. The dyeing of the syn-
thetic fibre, nylon, is instructive here.^^* This substance does not
provide much opportunity for the kind of reaction with acid dyes
that we have been considering, because an amino-group only
occurs at one end of the long chain-molecule. When this has reacted
by salt-formation with the anion of an acid dye, an increase in
acidity will result in a further, sudden uptake of dye, which is

?

=0
NH

Peptide group in nylon or protein

attributed to the activation of the peptide groups that occur re-
peatedly in the nylon chain.^^^ It is supposed that hydrogen ions
are taken up by the peptide groups, which thus become positively
charged and therefore attract dye-anions. If nylon can behave in
this way, there would not seem to be any reason why acid dyes
should not attach themselves similarly to the protein chain, in acid
solution. It is a striking fact that wool becomes softened ('ten-
dered') when it has taken up more than a certain amount of an
acid dye. This suggests an alteration in the main chain of the pro-
tein. A reaction of this sort would be unrelated to the preponder-

THE DIRECT ATTACHMENT OF DYES TO TISSUES I99

ance of basic or acidic side-groups, and the appearance under the
microscope would therefore necessarily be diffuse.

The electrostatic forces between oppositely-charged ions act
over much greater distances than the 'short-range' forces concerned
in covalent and hydrogen bonds. That such short-range forces may
be important in textile-dyeing has been especially stressed by
Neale.^^^ The process we have been considering will not account
for the dyeing of cotton, for this is a negatively-charged substance
that is generally dyed by acid dyes. The energy necessary to bring
the similarly-charged bodies together is provided by thermal
agitation. It is for this reason that high temperatures are used.

In microtechnique we are not faced with exactly this situation,
because we do not ordinarily dye negatively-charged objects with
the negatively-charged ions of ordinary acid dyes. Still, if dye-ions
can be attached to cotton bv forces other than those we have been
considering, the possibility exists that in microtechnical dyeing
long-range electrostatic forces merely play a part similar to that
played by thermal agitation in the dyeing of cotton. The close,
final attachment may then be achieved through hydrogen bonds.
A useful short summary of the various parts of dye-molecules that
could serve for hydrogen bonding to the hydroxyl groups of
cellulose is given by Evans. ^^^ Either the hydrogen or the oxygen of
these hydroxyl groups can participate in a hydrogen bond, the
former making a link (for instance) with the nitrogen atom of an
amino-group or one of the nitrogens of an azo-group of the dye,
the latter with a hydrogen of a -C=C- group. These reactions

H H
only occur so long as the hydroxyl groups of the cellulose are intact.
Bonding of this sort presumably takes place in microtechnique
when we use a direct cotton dye such as Congo red to colour
cellulose cell- walls.

It is not only cellulose, however, that can bind itself to dyes in
this way. The dyes commonly used to colour nylon (which are not
the ones mentioned above (p. 198) as colouring it from acid solu-
tion) appear to form hydrogen bonds with the peptide groups of
the fibre. If so, the amido-groups of the protein chain may also be
available for the attachment of dyes. A link could be formed
connecting the hydrogen of a peptide group forming part of a
protein chain to the nitrogen of an amino- or azo-group in a
dye.

2D0 DYEING

General intermolecular attraction (van der Waals forces) are
thought sometimes to aid the indiscriminate anchoring of dyes,
provided that close enough approximation to the substrate can
somehow be attained.

I

NH . . . NH,

A possible hydrogen bond (...) between a peptide group of a protein chain

and an amino-group of a dye

Although these kinds of bonding must be kept in mind, yet
there is at present no strong evidence that they play a dominant
role in microtechnical dyeing. The biologist has one advantage
over the textile chemist in judging the forces that bind a dye to its
substrate. The tissues of plants and animals, as studied in the
biological laboratory, provide us with very obvious visible indica-
tions as to whether a dye is acting in conformity with the electric
charge on its ions or not. It is true that the cortex of wool is some-
what acidophil and the medulla somew^hat basiphil; ^-^ but the
contrast is not very sharp, and anyhow textile chemists do not
devote a very great deal of study to the appearance of dyed wool
under the microscope. When a biologist looks at a microscopical
preparation of the most ordinary kind, every cell suggests the
supremacy of ionic forces, for the acidic chromatin is coloured by
the basic dye and the ground cytoplasm in a contrasting colour by
the acid dye.

If reactions between oppositely charged groups play a major
part in dyeing, one would expect a stoichiometric relation between
the amount of protein dyed and the maximum amount of dye that
could be taken up. This is a subject on which there is conflicting
evidence. One can measure the amount of inorganic acid with
which a particular protein will combine, and then compare this
with the amount of acid dye that can be taken up. It is claimed
that the number of arginine, lysine, and histidine groups in the
protein account for the amount of acid or acid dye that will com-
bine, and that there is therefore no reason to suppose that the
peptide groups of the main protein chain participate in the process
of dyeing. ^^^ Singer and Morrison, *^^ however, found that at pH 2

THE DIRECT ATTACHMENT OF DYES TO TISSUES 201

and 1 1 fibrin films took up far less acid and basic dyes respectively
than would be expected if all the charged groups of the protein in
fact reacted with dyes. The subject, however, is complicated. Dye
ions often aggregate (p. 238), especially at the low temperatures
used in the biological laboratory, and this would make stoichio-
metric proportions unlikely. Dyes ions with two or more charged
groups might not be able to use all of them to make attachment to
oppositely charged groups on the protein, because the latter might
be too far apart.

The elTect of inorganic salts takes up a good deal of space in
works on textile dyeing, but is of less direct interest to the biologist.
In dyeing cotton and other cellulose fibres with certain direct
cotton dyes, it is usual to add sodium chloride or sulphate to the
dye-bath, as this greatly aids the uptake of dye. The dye-ions of
these direct cotton dyes carry the same (negative) electric charge as
the cellulose. In general, inorganic salts help dyeing in those
particular cases in which the electric charges oppose it, apparently
by favouring the near approach that is necessary for dyeing by
close-range bonds. *'^ On the contrary, they interfere with the
linkage between a dye-ion and an oppositely-charged group in the
object to be dyed, and also lessen the activity of many dyes by
increasing their tendency to fiocculation and thus lowering their
capacity to diffuse. Salts should not be used as buffers for dyes
without regard for these facts.

Temperature affects dyeing in several ways.^^*' ^'^ High tem-
peratures increase the rate of diffusion of dye-ions and also reduce
any tendency they may have to aggregate into larger particles (p.
241), which would move more slowly and penetrate less easily.
High temperatures also loosen the covalent bonds that hold protein
chains together, and dissolve the disulphide links: thus the protein
becomes more easily permeable. At 100° C i hour may suffice
for wool to take up as much of a dye as it can hold; but if the
temperature be kept down to 20" C, 5 months may elapse before
equilibrium between wool and the same dye is reached. Tem-
perature does not have much effect, however, on the amount of
dye eventually taken up: rather more is taken up from cold solu-
tions, except in those cases in which a dye cannot enter at all in
the cold (p. 241). Most biological material is very much more
easily penetrated by dyes than wool is, and the temperature is only
raised above that of the laboratory when a dye has a special ten-
dency to fiocculation (e.g. azocarmine) or when a tissue-constituent

202 DYEING

is particularly close-textured and therefore difficult to penetrate
(e.g. mitochondria).

The effects of fixation on dyeing have already been mentioned
in chapters 5 and 6, under the headings of the eight primary
fixatives selected for separate description. A general review of the
subject will be given here.

These effects can be studied by experiments on proteins.
Seki ^^^ fixed small pieces of egg-white in various ways; he either
dyed them whole or made paraffin sections. Fibrin films are very
suitable for work of this sort; they are chemically uniform and
require no sectioning.*'^ The effects of fixation on the dyeing of
tissues has been investigated by several workers.^^^' ^^'^' *^^'*"^'^^
Whether proteins or tissues be used, the material may be exposed
to a mixture of a basic and an acid dye,^^^ or to basic and acid dyes
separately; the pH of the dye-solution may be controlled. ^^^
Instructions for carrying out simple experiments on the effects of
fixatives on dyeing are given in the Appendix (p. 325).

For studies of this kind it is important to avoid cells (such as
nerve-cells) that have much RNA in the cytoplasm, for this would
colour strongly with most basic dyes and therefore mask the effect
of fixatives on the reactions of dyes with the cytoplasmic proteins.
Strongly basic protoplasm, such as that of mammalian red blood-
corpuscles, is also unsuitable. One needs an example of typical
cytoplasm and typical chromatin. Seki *^^ chose the skin of the
frog and mammalian kidney. The convoluted tubules of the latter
organ and the spermatogonia and spermatocytes of mammals are
particularly suitable. ^^ The late spermatids and spermatozoa them-
selves are unsuitable not only because of the absence of unspecial-
ized cytoplasm, but also because of the basic nature of the nuclear
material.

Certain possible sources of error should be noted. The intensity
of dyeing is often different at different depths in a piece of tissue.
It is best to use small pieces of nearly uniform size and to compare
cells in the middle of each piece. Some fixatives shrink the cyto-
plasm strongly. In its shrunken condition it may give a false im-
pression of taking up a lot of dye, when in fact the same amount of
dye, spread over the cytoplasm of an unshrunken cell, would look
quite pale. It is desirable that quantitative methods should be
introduced into work of this kind. This has already been done in
studies made with fibrin films. *"^

THE DIRECT ATTACHMENT OF DYES TO TISSUES 203

Unfixed proteins are generally neither strongly basiphil nor
strongly acidophil. Chemical fixation generally increases their
colourability by dyes. This is well shown by fibrin films. ^''^'^'^
Fixation by heat has the same effect. ^^^ Certain fixatives favour the
action of basic dyes, others that of acid dyes; others again allow
easy coloration by both.

Formaldehyde favours basic dyes more than any other fixative
does. Mercuric chloride has the same tendency, but in a less
marked form. These facts are related to what is known of the re-
actions of these two fixatives with proteins, for in both cases there
is blocking of -NHg groups (pp. 53 and 59). Kelley ^^^ con-
siders that mercuric chloride favours acid dyes; he thinks the main
effect of the salt is to block carboxyl groups, while amino-groups
are left free for linkage wdth acid dyes. This is contrary to what
is known of the chemistry of fixation by mercuric chloride, and
also to the observable facts of dyeing.

From some of the remarks in the literature one might imagine
that osmium tetroxide almost abolishes the capacity of tissues to be
dyed. Even Seki,*^^ so reliable as a general rule, gives this impres-
sion. Actually this fixative leaves cytoplasm readily colourable by
basic dyes (after bleaching), though scarcely at all by acid ones.
Anyone can prove this for himself by carrying out the experiment
described in the Appendix (p. 325). Basic fuchsine gives particu-
larly intense coloration of the convoluted tubules of the mammalian
kidney. These facts cannot be correlated with what is known of the
reaction of osmium tetroxide with proteins (p. 62). From the
effects of dyes one would suppose that amino-groups were almost
eliminated, but carboxyl groups left free for linkage wdth basic
dyes. It must be mentioned that basic dye-lakes (p. 207) act less
strongly than basic dyes on tissues fixed by osmium tetroxide.

Ethanol is intermediate between such fixatives as formaldehyde,
mercuric chloride, and osmium tetroxide, which make cytoplasm
basiphil, and the ones to be mentioned below, which have the
reverse effect. It allows fairly easy dyeing of proteins and cyto-
plasm by both basic and acid dyes. This must be correlated with
the fact that it is a non-additive fixative, which would not be ex-
pected to have much effect on the proportions of the acidic and
basic groups available for linkage with dyes.

In sections of tissues fixed with acetic acid, the cytoplasm shows
a general resemblance in its reaction to dyes to that fixed by ethanol,
for both basic and acid dyes are taken up fairly readily.

2^4 DYEING

The fixatives that particularly favour acid dyes are trichloracetic
acid,*^^ picric acid, and chromium trioxide. Seki ^^^ w^ould add
potassium dichromate to these, but after this substance has been
allowed to act on the mammalian kidney, the cytoplasm of the
convoluted tubules is readily coloured by basic dyes.^^ The facts
suggest that trichloracetic acid, picric acid, and chromium trioxide
tend to block carboxyl groups and leave amino-groups free for
linkage with acid dyes. Seki, however, considers that basic proteins
are immobilized by the alkaloidal reagents and remain in the
tissues to react with acid dyes, while acidic proteins simply
dissolve away. Unfortunately we have not nearly so much know-
ledge about the reactions of these substances with proteins as
we have about the reactions of formaldehyde and mercuric
chloride.

The effects of fixatives in blocking carboxyl or amino-groups
are reflected in changes in the iso-electric points of proteins. Such
changes are most readily observable with fibrin films, which can be
subjected to direct cataphoresis.^'^ The iso-electric point of un-
fixed fibrin is pH 6-0 ; denaturation by heating (i min. at ioo° C)
lowers this figure to 5-7; fixation by formaldehyde (10%, 16 hr.) to
5-2. Estimations of the shift of the iso-electric point of tissue-
constituents can be made by experiments with dyes used at
particular pHs.^^^' ^^^ It is found that various objects in the tissues
can be coloured by basic dyes at a lower pH after fixation by for-
maldehyde than after any other fixative. Ethanol has been found to
have little effect on the position of the iso-electric points of tissue-
constituents.

The dyeing of chromatin is more complicated than that of the
cytoplasm. It is likely that fixatives generally split DNA from
protein, and the colouring is then mainly that of DNA by the
basic dye.^^^ A virtue of acetic acid is that it leaves chromatin
scarcely colourable by acid dyes, so that basic dyes are not masked.
Many other fixatives allow strong coloration of chromatin by basic
dyes: for instance, mercuric chloride, formaldehyde, ethanol, and
potassium dichromate. The two latter, however, do not coagulate
nucleoproteins, and there is therefore no immobilization of
chromatin in its original sites. When it is contained in the nucleus,
however, it is prevented from escape by the nuclear membrane, and
it distributes itself almost at random within the nucleus. This is
particularly obvious when one examines the first meiotic prophase
in a gonad fixed in ethanol or potassium dichromate. No one would

THE DIRECT ATTACHMENT OF DYES TO TISSUES 205

choose either of these fixatives for work on chromosomes, except in
mixtures.

Despite statements to the contrary, osmium tetroxide allows
quite strong colouring of chromatin by basic dyes; but it often
makes the nuclear sap basiphil, and this clouds the structure of
the nucleus. One would not use this fixative alone in studies of
prophase or telophase changes, or in any other investigation re-
quiring a glassy nuclear sap.

The reactions of picric acid with chromatin are very curious. ^^
The protein part is coagulated, but the DNA is set free and dis-
solves (compare Levene ^^^). The chromatin is now represented
only by protein, and the nature of the latter determines the reaction
with dyes. Since the protein component of chromatin is seldom
strongly acidic, there is little affinity for basic dyes; but if it
chances to be definitely basic (as, for instance, in the spermato-
cytes of mammals), acid dyes will be taken up strongly. We thus
have the curious spectacle of meiotic chromosomes strongly dyed
by acid dyes, but feebly by basic ones.

It is not unusual for the protein of chromatin to be colourable
by acid dyes, after DNA has been separated off by the action of
fixatives. Acid dyes are used to colour chromatin in certain tech-
niques (acid fuchsine in Mallory's tricolour method ^-^ and azo-
carmine in Heidenhain's 'Azan').^^^ After most fixatives the DNA
will still be present, but will not mask the colouring of the protein
by an acid dye unless a basic dye is used as well. The acid dye
works more strongly, however, if DNA-ase is allowed to act fi^rst.

In the chromatin of the spermatozoa of certain fishes the DNA
is combined with protamine. The strongly basic nature of this
simple protein, caused largely by its high arginine content, pre-
dominates to such an extent that the substance as a whole, with its
DNA, is highly acidophil and scarcely takes basic dyes, whatever
fixative may have been used.

If a particular fixative leaves some of the phosphoric groups of
DNA still combined with protein, this will necessarily result in a
low affinity for basic dyes.

The reason why formaldehyde interferes with the dyeing
of chromatin by borax-carmine follows from the foregoing con-
siderations. In alkaline solution carminic acid is an acid dye, with-
out affinity for DNA. It can combine, however, with the basic
groups of the protein of nucleoprotein, and then, when the tissue is
subsequently acidified and it becomes a basic dye, it can fix itself to

206 DYEING

the DNA. If, however, formaldehyde was used as fixative, the
protein of the chromatin will have been rendered acidic and there-
fore will have taken up little carmine from alkaline solution ; there
will consequently be little dye present in the chromatin to link up
with DNA on subsequent acidification.

In routine preparations we usually want to dye the chromatin
in one colour with a basic dye (or basic dye-lake, p. 207) and the
cytoplasm in a contrasting colour with an acid dye. What does this
involve? Ideally, the DNA must be struck off by the fixative from
the protein with which it was combined, and precipitated instantly
without change of position in a form in w^hich it will combine
readily with basic dyes ; the protein of the chromatin must have a
low affinity for acid dyes, so that the colour given by the basic dye
shall not be masked ; and the cytoplasm must have little affinity for
basic dyes, but link strongly with acid ones. The fixative that
leaves tissues most nearly in this condition is chromium trioxide,
though chromatin fixed by this substance has not so strong an
affinity for basic dyes as one would wish, and retains some affinity
for acid ones. Acidified potassium dichromate acts similarly to
chromium trioxide. (See p. 132.)

Many fixative mixtures probably owe their continued popularity
largely to the fact that they happen to leave the iso-electric points
of the tissue-constituents in convenient positions; that is to say, in
positions in which it is easy, without troubling to use buffers, to
colour the chromatin with a basic dye and the ground cytoplasm
with an acid dye of contrasting colour. Some shift of the iso-
electric point towards higher pH would be helpful towards this
end, w^hich is realized by such ffuids as that of Zenker. ^^* If this
shift did not occur, ground cytoplasm would generally be too
basiphil to give good colour-contrast with chromatin.

It is a special property of mercuric chloride that it leaves tissues
in a state in which they are readily coloured by dyes of all sorts (but
particularly by basic dyes and dye-lakes). No adequate explanation
of this familiar fact has been provided. One must suppose that the
tissues are fixed in such a form that they are readily penetrated by
large ions, and that many acidic and basic groups (especially the
former) are available for linkage with dyes.

CHAPTER I I

The Indirect Attachment of Dyes

to Tissues

It is characteristic of dyes that when they are dissoh^ed in water
or a mixture of w^ater and alcohol, they attach themselves directly
to certain tissue-constituents in one or more of the ways described
in the last chapter. Some of them, however, have an alternative
method of attachment, involving the presence of another substance
besides dye and solvent; and when this substance is present, their
behaviour is quite different. Great advantage can sometimes be
taken of this difference in behaviour, both in the textile industry
and in microtechnique. A dye that is almost useless in simple
solution may become of major importance.

The salts of certain metals are the chief substances that radically
alter the behaviour of particular dyes. These salts were called
mordants because they were thought to bite into certain textile-
fibres and thus give attachment to dyes that would not work
satisfactorily by themselves. The word was taken into our language
from French.

A mordant is capable of entering into chemical combination
with a dye. The resulting substance is called a lake. This word
had a curious origin. It derives from lac, a Hindustani word mean-
ing the waxy material produced by the scale-insect, Tachardia
lacca. The females of this species attach themselves in great
numbers to the twigs of certain Indian trees and produce an
abundance of waxy material, which fills up all the spaces between
them. The waxy material is sold as lac, or, if in flat plates, as
shellac. The insects themselves contain a large quantity of lac-dye,
which is chemically related to carminic acid; ^^^ this is said to be
stored chiefly in the ovaries. ^^ The dye is dissolved out and then
precipitated by potassium alum. The name lac became attached
not only to the wax but also to the precipitated dye, and afterwards
to the product of the cochineal insect precipitated in the same way
('crimson lake'). The alum plays a double role. It precipitates the

207

20S DYEING

protein fraction of the crude dye, and also combines with at least
part of the true dyestuff.

In a valuable work on dyeing published in 1813/^ it was sug-
gested that dyes used with mordants should be called 'adjective'
dyes; those used without mordants, 'substantive'. These words are
still used, but it must be remembered that there are not really two
classes of dyes. It is true that many dyes cannot be used adjec-
tively; but dyes that can be so used wdll also colour biological
material substantively.

The great advantage of the use of mordants in microtechnique is
that when once the tissue/mordant/dye complex has been formed,
it is insoluble in all the neutral fluids ordinarily used, so that
subsequent colouring with other dyes is easy and there is no hurry
in dehydration. The lakes are basic in action. Their fastness to
alcohol is a very great advantage over ordinary basic dyes and
renders them particularly suitable for the colouring of whole
mounts, which cannot be dehydrated quickly. Certain dyes, par-
ticularly haematein, produce w^eak, indefinite, or unsuitable colours
w^hen used substantively, but give brilliant or intense results when
used with a mordant.

In the textile industry the mordant is sometimes used first and
the dye afterwards (two-bath method); sometimes the two are
used together (single-bath method) ; sometimes the dye is used
first and the mordant afterw^ards ('afterchrome' method). '^^ In
microtechnique the mordant is very seldom used after the dye,
though two examples can be quoted. ^'^^' ^^^ The other methods are
both in common use.

Two problems at once present themselves. How does the mor-
dant attach itself to the tissue ? How does the dye attach itself to
the mordant (or, in other w^ords, what is the lake)?

In trying to answer these questions one gets far less help from
the textile chemists than might be expected. There are several
reasons for this. In microtechnique the most important dyes used
adjectively are haematein and carmine, but in the textile industry
the great majority of mordant dyes belong to the azo-group. No
azo dye is used with a mordant in ordinary microtechnique. A far
more important reason, how^ever, concerns the nature of the
mordants used. The industrial dyer uses chromium mordants
almost exclusively nowadays, but these are much less used in
microtechnique than iron and aluminium. T'his would perhaps
not matter very much in itself, for knowledge gained about the

THE INDIRECT ATTACHMENT OF DYES TO TISSUES 209

action of chromium might be apphcable to the other metals; but
unfortunately the ordinary use of chromium in industry is radic-
ally different, for it is applied as sodium or potassium dichromate,
that is to say in an anionic complex, whereas in microtechnique we
use the metals as cations. It is an extraordinary misfortune that
in fixation we cannot profit from industrial chemistry" because we
use anionic chromium while the tanner uses cationic, and in dyeing
we cannot profit much because we use cationic chromium while the
professional dyer generally uses anionic.

In short, in microtechnique w^e generally use iron, aluminium,
and cationic chromium to mordant for haematein, carmine, and
certain oxazine dyes, while the industrial dyer generally uses
anionic chromium to mordant for azo dyes.

The chemistry of mordanting in industry has been thoroughly
investigated and must be mentioned very shortly here.^^^' ^^*^' ^^-' "^
The acid azo dye commonly has -OH groups ortho to the azo-
group. On reaction with the mordant at high temperature, the

_OH HO_ _0^t 0_

\_/^ IN x^_^ ^_^ IN IN ^^^

Skeleton fortnulae of an azo dye before and after linkage with chromium

chromium atom makes covalent links with the oxygens of the
hydroxyl groups and obtains a dative covalency from one of the
nitrogens, at the same time showing a negative electric charge.
It follows that the mordant dye complex has the character of an
acid dye, which can react with wool by making salt-linkages with
amino-groups of the protein. The dye generally possesses sul-
phonic groups (not shown in the skeleton formula), which again
are acidic and can react in the same way.^^^ Thus the mordant/dye
complex reacts with positively-charged groups in the wool, or
indeed may even be held in place by the mere fact that it forms too
large a particle to escape from the pores of the wool.^^^ There is
strong evidence that one chromium atom reacts with two mole-
cules of the dye,*^^ but this is not represented in the simplified
formula shown.

Long and complicated researches have led to the conclusion that
the mordant/dye complex of the textile dyer is to be regarded as
acting as though it were an acid dye, but in microtechnique we can
generally tell whether any colouring agent is acting as an acid or a
basic dye by a momentary glance down the microscope. The fact is
o

210 DYEING

that most tissues of plants and animals are extremely unlike wool.
An acidic lake would be useless to us (see p. 223): our mordant
colours act as basic dyes, and are indispensable.

The chemistry of mordanting in microtechnique is perhaps the
most perplexing of all the subjects with which this book deals.
The literature is scattered among journals seldom read by any
one person, and no serious attempt has ever previously been
made to integrate our knowledge of the subject into a compre-
hensible whole. The most convenient arrangement for the reader
would be to start with a detailed description of the w^hole process
of attaching one particular dye wdth one particular mordant, and a
facile story of this kind could indeed be written. In fact, however,
we lack the knowledge necessary for a consecutive account of the
whole process. Ionization of mordants has been best described
with the salts of chromium, lake-formation with those of alu-
minium, and the attachment of metal to tissue wdth ferric salts:
different dyes must be chosen to illustrate particular points. In
what follows no attempt will be made to push generalization too
far. It is better at the present time to disclose the gaps in our know-
ledge than to put forward a consistent theory too confidently. Certain
important facts and the rough outline of a synthesis will emerge.

The mordants commonly used in microtechnique are salts,
especially sulphates, of iron, aluminium, and chromium. Double sul-
phates or alums are generally used. The use of alums rather than
simple sulphates is perhaps due in part to historical causes. Alums
are easily crystallized and therefore easily purified from the rocks
that contain them. Their use in dyeing has been known from rather
remote times. ^^ Until about the middle of the fifteenth century the
only rocks known to contain a suitable mordant were in Turkey;
but when Constantinople had been captured by the Turks in
1453, a fragment of the knowledge that escaped with the refugees
to the western world concerned the recognition of suitable rocks
and the mode of preparation of the mordant. Alum was first mined
in Great Britain about the end of the reign of Queen Elizabeth I.

The alums generally used are these: —

potassium alum, Al2(S04)3.K2S04.24H20;
ammonium alum, Al2(S04)3.(NH4)2S04.24H30;
iron alum, Fe2(S04)3.(NH4)2S04.24H20;
chrome alum, Cr2(S04)3.K2S04.24H20.

THE INDIRECT ATTACHMENT OF DYES TO TISSUES 211

The formulae can also be written as AlK(S04)2*i2H20, etc.,
but this method shows the composition less well. There is no
purpose in constructing the formula in a way that will make the
molecule appear as small as possible, since there are no molecules
in the crystal, and only ions in solution.

Aluminium, ferric, and chromic sulphate act similarly to the
alums. There is no compelling reason why sulphates should be
used in preference to other salts. Ferric chloride, for instance, has
been used as a mordant for haematein.^^^

The cation of the mordant salt, then, is the part that interests
us in microtechnique. The metal is in each case tervalent in these
compounds, but the ions are complex. The water of crystallization
is partly bound up with the metal. For instance, the ferric cation
that gives the violet colour to a crystal of iron alum is
[Fe(OH2)6]^^^. The chromic cation makes a crystal of chrome
alum almost black. When a brilliant light is shone through a small
crystal, the colour is seen to be violet. The red component is
easily seen when a small crystal is held against an electric light
bulb. The cation responsible for the deep violet colour is
[Cr(OH2)6]^^^. The water molecules are co-ordinated to the iron
or chromium atoms by dative covalent bonds from their oxygen
atoms. The metal may be described as sexi-covalent.

-HaO^ ^OHa 1 + + +

HaOCr^OHs

.HsO^ ^OH2_j
The chromic cation in a crystal of chrome alum

When iron alum is dissolved in water, the solution is not violet
but yellow or brownish-yellow. When chrome alum is dissolved
in hot water, the solution is not violet but green. In both cases
the solutions are quite strongly acid. The changes that occur in
the cation have been carefully studied in the case of chromium. A

-U.O^yOU -| + +
H2O— Cr— OH2

The chromic cation in a solution of chroyne alum

hydrogen ion is lost from the cationic complex and goes off with
one of the positive electric charges, thus acidifying the solution,
and a second may follow suit.^^^ Similar changes occur when iron
alum dissolves, though the yellowish cationic complexes produced
are usually represented as [FeOH]" ' and [Fe(0H)2]^.

212 DYEING

Aluminium behaves similarly, but both the simple sulphate and
potassium alum are colourless, and there is no production of colour
to call attention to the change in chemical composition of the
cation on solution. The cationic complexes in solution are usually
represented as [A10H]^+ and [A1(0H)2]^

The production of hydrogen ions in the course of these changes
results in varying degrees of acidity. The following figures relate
to solutions of the crystalline salts (ordinary laboratory chemicals,
as used in microtechnique): —

pH

chrome alum, 5% . . . . .1*8

iron alum, 5% . . . . . • i'9

,, 2*5 /o • • • • . 2'I

potassium alum, 5% ..... 3-2

The complex cations that we have been discussing, in solutions
rendered acid by the act of dissolving, are the essential part of the
mordant — the part that must react with the dye and with the tissue,
so as to leave a link between them. As we have seen, the attach-
ment with the dye can occur either before or after the mordant
metal has fixed itself to the tissue. It will be convenient to consider
first the nature of a soluble lake as used in the single-bath process;
that is to say, we shall forget the tissue for the moment and think
only of the reaction of the dye with the mordant.

Dyes that form lakes possess a phenolic -OH group, which
plays an important part in lake-formation. Sodium hydroxide will
react with phenol to form sodium phenolate and water, and ferric

?

Na
Sodium phenolate

iron will react in a comparable way. A much firmer bond can be
made between certain phenols and iron, however, because they
can provide a second link with the metal. For instance, salicylic
acid reacts with iron in the way just described, the metal replacing
the hydrogen of the phenolic -OH group. ^^^ This happens when
salicylic acid is treated with ferric chloride. This, however, is not
all that happens. ^^^ The organic acid has an oxygen atom con-

THE INDIRECT ATTACHMENT OF DYES TO TISSUES 213

veniently situated to donate electrons to an iron atom that has re-
placed the hydrogen of the -OH group. Thus a dative covalency is
formed, as is shown in the formula by an arrow, and the iron atom

HO— C I HO— C

II '
O OH

Salicylic acid Salicylic acid combined zvith iron

is now gripped by a pair of pincers. It wdll be noticed that the iron
atom now forms part of a six-membered ring, the atoms being in
this order: — iron, oxygen, carbon, carbon, carbon, oxygen.
Actually the iron atom is gripped by three pairs of pincers (though
this fact is not shown in the formula), for the iron atom can
replace hydrogen in three molecules of salicylic acid, each of
which can form a second bond with the iron through an oxygen
of its carboxyl group.

To achieve this result, one must have a bi- or multivalent metal
that is capable of forming covalent bonds, and an organic com-
pound that possesses both a hydrogen atom replaceable by the
metal and also a donor atom (here oxygen), capable of donating
electrons to the metal to form a second link. These are exactly the
conditions that are fulfilled when a lake is made. This fact was
first realized nearly half a century ago by the celebrated Swiss
chemist, Werner, in 1908,^^^ though naturally he did not express
himself in terms of the electronic theory of valency. He realized
that the mordants were salts of metals that could form innere
Komplexsalze, and he quoted [Cr(OH2)6]X3 (X standing for the
anion). The fact that chromium is capable of receiving dative
bonds is implied by the formula he wrote, for, as we have seen, six
molecules of water are co-ordinated to the metal through their
oxygen atoms. Werner expressed himself thus: — 'From the re-
ported results of the experiments it can no longer be doubtful
that the property of chemical compounds to connect wdth mordants
depends upon the property of the latter to form internal metal-
complex salts.' ^^^ Of the other partner to the transaction he wTOte,
'Dyes that connect with mordants are hereafter constitutionally
characterized by the fact that a salt-forming group and a group
capable of producing a co-ordinate bond with the metal atom are
so arranged that an internal metal-complex salt can originate'. ^^^

214 DYEING

It is fitting that the man who was afterwards to receive the Nobel
Prize for his co-ordination theory of valency was the first person to
grasp the essentials of lake-formation.

The chemistry of this process was subsequently studied by two
British chemists, Morgan and Smith, ^^^ to whom we owe the
expressive term 'chelate' for the pincer-like grip in which a dye
holds the mordant metal. They took the word from the analogy
with the chela of a lobster.

It now remains to look for the phenolic -OH group and nearby
donor oxygen in the dyes that form lakes. Alizarine provides a very
simple example. A phenolic -OH is close beside a suitable oxygen
atom, and a six-membered ring (aluminium, oxygen, carbon, car-

OH

Alizarine Alizarine aluminium lake

bon, carbon, oxygen) is readily formed. (The aluminium atom is
capable of linking with three alizarine molecules.) One has only
to look at the formula for carminic acid on p. 177 to see that it is
capable of acting in exactly the same way.

It is important to notice that the metal atom (whether alumin-
ium, chromium, or ferric iron) makes two different kinds of links
with the dye. These links may for convenience be called primary
and secondary. The primary link is made by the substitution of
the metal for a hydrogen atom in an acidic -OH group. It is
reasonable to suppose that this primary linkage is initially electro-
valent, though it may be replaced by a somewhat polar covalency.
The secondary link is the dative covalency with the electron-
donor oxygen atom. The cation could make room for such a
covalency by losing one of the -OHg or -OH groups held to the
metal by 'subsidiary' valencies. (See the formulae for chromic
cations on p. 211.)

It was pointed out nearly 70 years ago^^* that many anthra-
quinone dyes that can be used with mordants have two phenolic
-OH groups in or//zo-relationship to one another, and it was at first
thought that these gripped the metal. The action of the second

THE INDIRECT ATTACHMENT OF DYES TO TISSUES 215

-OH group is not clear,^^^ and indeed it is absent in lac-dye,
which works with mordants.

In the absence of a nearby donor oxygen atom, two -OH groups
can form chelate compounds with iron. Certain derivatives of
catechol provide examples of this.^^^ It is therefore interesting to

HO

HO^I

\/
Catechol

find two -OH groups in this position in haematein (p. 173), which,
however, also possesses a phenolic -OH in close proximity to a
donor oxygen, in another part of the molecule.

The oxazine dyes that work with mordants also possess the
necessary groupings for the formation of chelate bonds with

OH ^

(H3C)YY'/Y

\0H

Gallocyanine

metals, though perhaps in a somewhat disguised form. The
formula for gallocyanine is printed here in such a way as to em-
phasize the relationship with other mordant dyes.

Haematein, carminic acid, and the mordant oxazines are all
amphoteric dyes. Indeed, one would suspect this from a glance at
their chemical formulae. Gallamine blue, for instance, presents two
basic groups (-NHg and -N(CH3)2) and two acidic (-0H). The
iso-electric point of haematein is about pH 6-5, of carminic acid
about pH 4-2, of gallocyanine and gallamine blue about pH 4-1.
The lakes are in all cases basic throughout the pH range in which
they are used, and they act like basic dyes apart from their com-
plete insolubility in neutral fluids after they have once attached
themselves to the tissues. The positive charge on the lakes can be
proved by cataphoretic experiments.*^^' ^^^

Sometimes a dye-lake has a colour that is different from that of
its parent dye. Thus haematein is yellowish at about pH i and
changes its colour towards orange and dirty red as aciditv lessens
towards neutrality ; but the aluminium lake is blue and the charac-
teristic iron lake dark blue or blue-black. Carminic acid is some-
what less affected, for the dye by itself, whether in acid or alkaline

2l6 DYEING

solution, is red, and the aluminium lake crimson. The oxazine dyes
are still less affected in colour by combination with mordants.

The oxazine dyes do not generally work well if applied in simple
solution after mordanting ^^^ and the single-bath method is
commonly used with them. Carminic acid forms very stable
soluble lakes, and here again the single-bath method is appro-
priate. Haematein can be used as a soluble lake, but the two-bath
method is generally used when iron is the mordant for this dye.
In Hansen's so-called Trioxyhdmatein ^^^ the dye is mixed with
the iron mordant, but precipitation is liable to occur unless pre-
cautions are taken. It was Ehrlich ^^^ who first overcame the
instability of the aluminium haematein lake, by the addition of
acetic acid. The effect is to keep the dye separate from the mordant.
The acidified solution shows the reddish colour of the dye, not the
blue of the lake. How the acid acts is not perfectly clear. The
mordants themselves, as we have seen (p. 212), are themselves
strongly acid. It must be presumed that the additional hydrogen
ions stop the loss of hydrogen from the phenolic -OH group of the
dye, and thus prevent its replacement by the metal of the mordant.
(See p. 221.)

Strong acid is needed to prevent the formation of an iron lake
or break it when once formed. Sulphuric acid may be added at
2% to prevent the precipitation of the iron lake of coelestine
blue.^^^ Since this lake has the same colour as the dye, the course
of events is not so easy to follow as when aluminium and haematein
are kept apart by acidity and then allowed to join by neutralization.
With iron and coelestine blue the tissue/mordant/dye complex can
be formed without removal of the acid. The presence of tissue
seems to favour the formation of the lake.

It is a curious fact that some lakes have greater powers of
penetration than their parent dyes. Thus gallamine blue diffuses
into gelatine more slowly than its lake with chrome alum.^^^ It is
evident that the dye by itself is somewhat aggregated, and that the
mordant disperses it.

We turn now to the attachment of the metal to the tissue.

It is desirable to say at the outset that the metal will eventually
be held to the tissues by bonds that are very similar to those that
hold it to the dye. The primary linkage is with acidic groups
in the tissues, and this is the reason why dye-lakes act as though
they were basic dyes.

THE INDIRECT ATTACHMENT OF DYES TO TISSUES 217

The attachment of metal to biological material has been
specially studied by Wigglesworth.^^^ Sections of tissues fixed in
various routine fixatives were placed in solutions of iron alum
and then washed in water; the places where the metal had become
attached were made visible by treating the sections with ammo-
nium sulphide and thus producing dark sulphide of iron. A careful
survey of the resulting slides suggests strongly that the metal is
taken up by the acidic groups in the tissue, notably the phosphoric

groups of the nucleic acids and the — C/ groups of proteins.

The chromatin (especially that of chromosomes) is darkened.
This is partly the result of their nucleic acid content; but w^hen
the nucleic acids have been removed by treatment with hot tri-
chloracetic acid, the colour still develops by reaction of the iron
with the protein component of the nucleoprotein.

The reaction with different proteins is illuminating. Thus
myosine, which contains many acidic groups, is strongly darkened,
while salmine, which lacks such groups, is scarcely touched ; other
substances, acidic in varying degrees, show, with one or two
exceptions, a degree of darkening proportional to their acidity.

If the carboxyl groups in the proteins are methylated by pro-
longed immersion in acidified methyl alcohol, the uptake of iron
is much reduced. Deamination by formaldehyde has, on the con-
trary, no such effect, and it is thus clear that the -NHg groups are
not concerned. ^^^

Iron is taken up fairly strongly by the proteins of ground cyto-
plasm, but not nearly so strongly as by chromatin; also, in certain
circumstances, by elastin (p. 233); also, though feebly, by collagen.
Iron haematein colours these tissue-constituents brown, in con-
trast to the black or blue-black of chromatin. Mollendorff ^^^
claimed that iron haematein gave black when it acted as a pre-
cipitant dye, and brown when it permeated an object evenly
(p. 197). The two contrasting colours resemble those produced
when ammonium sulphide is substituted for haematein. ^^^ A
completely satisfactory explanation of these facts has not yet been
given.

There can be little doubt that iron is also bound by the phos-
phoric groups of phospholipids, if the fixation and after-treatment
have been adapted to the retention of these substances. It has been
shown that after all the RNA has been removed from the tissues by

2l8 DYEING

ribonuclease, the mitochondria are still colourable by iron
haematein; ^'^^ this has been attributed to their lipoprotein con-
tent. ^^^ Other acidic lipids besides phospholipids may perhaps
also bind iron, but the idea that iron haematein attaches itself to
nothing in cells except acidic lipids ^^^ is untenable.

Presumably the cationic iron complex first makes a salt-linkage
with the available acidic groups, but the characteristic reactions of
ferric iron are not given and it is thought that a non-ionizing
complex is formed with adjacent carboxyl and hydroxyl groups. ^^^

O
O

NH

I
HC(CH2)2C

c=o

I
NH

"?
f

Fe

CH2( >0

\— /H

Diagrammatic representation of a possible boyiding of iron with glutamic acid
atid tyrosine residues in a protein chain. {The zchole of the iron complex is not
shown.)

A theoretically possible arrangement in conformity with this
view is shown here. Two amino-acids of a protein are seen to be
holding the iron in their grip. Similar bonding could take place
with nucleic acids, through a sugar hydroxyl and a phosphoric
group.

When iron has been taken up by the tissues, the compound into
which it has entered is readily broken down by ammonium sul-
phide and it appears once more in an ionic form. Everything is at
first coloured blue or blue-grey; but when the section is washed
in water and thus exposed to atmospheric oxygen, an interesting
differentiation occurs. Certain parts, such as the chromatin,
remain blue or bluish, while others, such as collagen and the
contractile substance of muscle, become brown. It is thought that
the iron is ferrous in the one case, ferric in the other. At first
the iron appears everywhere as ferrous sulphide, and remains
as such wherever there are reducers (such as sulphydryl groups)
in the proteins or other cellular constituents; but where there
are not, oxidation can occur, with development of a brown
colour. ^'^^

THE INDIRECT ATTACHMENT OF DYES TO TISSUES 219

There is a contrast between the uptake of iron on one hand, and
of chromium and aluminium on the other. The two latter metals
attach themselves only slightly to the proteins of the cytoplasm,
and scarcely at all to elastin.^^^ They also have much less tendency
than iron to attach themselves to lipids (though anionic chromium
makes bonds with these; see pp. 107, 128). It is for these reasons
that we choose iron haematein to show mitochondria and certain
other cytoplasmic inclusions, but aluminium haematein or chrome
oxazine when we want to colour chromatin and little else.

The attachment of the chromic cation to tissue-constituents has
not been studied in detail. We have some information about its
behaviour in the dyeing of textiles, although, as was mentioned
above, most of the research on the chemistry of mordanting
has been done with anionic chromium. It is thought that the
attachment is by covalent linkages. Various groups in the protein
of wool are thought to displace water or OH or both from their
attachment to chromium in the cationic chrome complex, and to
co-ordinate with the metal in their place. The hydroxyl, amino-,
and amido-groups of the protein are mentioned in this con-
nexion. ^^^ It is also claimed that where two protein chains are held
together by an -S-S- bond, this may be split apart with produc-
tion of two -SH groups, and these, reacting with the chromium
atom, may cause the latter to act as a new link between the protein
chains. ^^2 It will be remembered, how^ever, that the dyeing of wool
is carried out at high temperatures.

In biological preparations it is certain that the phosphoric
groups of the nucleic acids and the acidic groups of certain muco-
substances make attachment with the chromium mordant more
readily than proteins, especially at low pH,^^^ and this also applies
to aluminium.

It will be noticed that the attachment of mordants to tissues is a
very complicated process, of which we have as yet only an imper-
fect understanding. The statement is commonly made in chemical
textbooks that the mordant metals are deposited in textile fibres as
gelatinous, insoluble hydroxides. This is not only an extreme over-
simplification, but quite untrue. Neither in textiles nor in ordinary
biological material are the metals deposited in this way. Had the
statement been true, there could be no question of that delicately
differential tying up of the metals with particular tissue-constitu-
ents that makes mordants so invaluable in microtechnique.
There is no gelatinous mass pervading our sections. If there had

220 DYEING

been, its combination with a dye would have rendered micro-
scopical study impossible.

Lakes can be used progressively. Hansen's iron haematein and
the oxazine lakes are ordinarily used in this way. It is, however,
almost invariable to work regressively when mordant and dye are
applied separately.

There are three separate methods by which differentiation can
be carried out when mordant-dyes are used regressively. The
agents used are mordants, acids, and oxidizing agents.

The tissue/mordant/dye complex is broken by excess of the
mordant. The dye distributes itself partly as a soluble lake with
the free mordant and partly as a component of the insoluble
complex; and since the amount of mordant in the complex is
small in comparison with the amount in the differentiating fluid,
nearly all of the dye will eventually associate itself with the latter,
if enough time be allowed. Since there will be much dye in some
tissue constituents (the chromosomes, for instance) and less in
others (such as the cytoplasm), a particular degree of differentia-
tion will leave no visible dye in certain parts though others remain
strongly coloured.

Since a mordant will continue to extract dye even when it has
already taken up a certain amount of it, but will colour tissues
powerfully if it is heavily laden with dye, there must be an inter-
mediate amount of dye that will cause strong colouring of basiphil
objects but not of those that have less affinity for the lake. This ex-
plains the facility with which certain lakes can be used. Grenacher's
alum carmine ^^^ and Mayer's carmalum ^^^ may be quoted as
familiar examples. Chromatin is coloured, but cytoplasm has little
tendency to take up the lake. As a result the exact period of dyeing
is unimportant. These dyes are particularly suitable for whole
mounts. Since too strong coloration does not occur easily, the
outer parts of a piece of tissue are not dyed very differently from
the interior.

It is interesting to notice the relative amounts of metal and dye
in such fluids as these. The weight of potassium alum crystals that
contains one atomic weight in grams of aluminium is almost
exactly equal to one gram molecular weight of carmine. Therefore,
if one wished to have a solution with 3 times as many dye
molecules as aluminium atoms, so as to combine as much dye as is
theoretically possible with the metal, one would take 3 g of car-

THE INDIRECT ATTACHMENT OF DYES TO TISSUES 221

minic acid to i g of alum crystals. Now Mayer's carmalum only
contains o-i g of carminic acid to i g of alum crystals.

If the amount of potassium alum in Mayer's carmalum is re-
duced by one-half, precipitation eventually occurs. It is evident
that a dye-lake only remains in permanent solution if the mordant
is present in great excess.

When a piece of tissue is mordanted with iron alum and then
treated with haematein, elastic fibres are not coloured. When,
however, the dyed tissue is put once more into iron alum, these
fibres take up the colour.^^^ This shows that during differentiation
by the mordant a soluble lake is formed, which is capable in certain
circumstances of dyeing, and also that the dye-lake penetrates the
elastic fibres more easily than its two components separately.

The differentiation of mordant-dyes by acids is more compli-
cated than that by mordants. Both the links in the tissue/mordant/
dye complex may be attacked. When a dye has a different colour
from its lake, it is easy to see that acidity undoes the bond between
mordant and dye. Aluminium haematein, for instance, is blue; but
when the lake is undone by the addition of a little acetic acid (as in
Ehrlich's haematein, ^^^ for instance), the colour of the dye itself is
seen. It must be supposed that the hydroxyl group of the dye,
which lost a hydrogen ion to form one-half of the chela that holds
the metal in its pincer-grip, is reconstituted in strongly acid
solution. The anion of the differentiating acid also plays a part, for
it must be one that does not make an insoluble salt by reaction
with the released cationic complex. Acetic and hydrochloric acids
are generally used. Neither of these forms an insoluble salt with
iron or aluminium.

The hydrogen ions of the differentiating acid presumably tend
to reconstitute, in unionized form, those acidic groups in the tissue
(phosphoric, carboxyl, hydroxyl, etc.) that were ionized when they
made their first contact with the cationic metal complex. The re-
actions, however, must be more complex than these few words
suggest. The bonds of the metal with the dye and with the tissue
are not, in their final forms, electrovalent. Further, it must be re-
membered that the mordant solutions themselves, in the absence
of added acid, have already quite a low pH (p. 212), and dye-lakes
are nevertheless taken up by the tissues from such solutions. The
addition of weak acids to mordant solutions does not have a big
effect on pH. For instance, 5% potassium alum shows a pH of 3-2 ;
if 3% of glacial acetic acid is added, the figure only falls to 2*45;

222 DYEING

if 5%, to 2-3. Now 5% chrome alum and 5% iron alum by them-
selves (p. 212) are considerably more acid than 5% potassium
alum acidified in this way; yet their low pH does not prevent
dyeing.

Another problem is presented by the peculiar resistance of the
tissue/iron/haematein complex to acidity. Quite strong acid must
be used to break this bond. This fact is very useful in microtech-
nique, for we can use weak acids in subsequent procedures without
removing the lake from chromatin ; but the reason for the difference
from other lakes is not obvious. We have not yet a full explanation
of the action of acids in loosening the tissue/mordant and mordant/
dye bonds that are formed in the ordinary processes of micro-
technique. It is probable that we should have had a better insight
into it if there had been any process corresponding to differentia-
tion in the dyeing of textile fibres. In industry, however, there
would be no purpose in trying to colour one part of one cell (in
wool, for instance) with a dye, while leaving another part colour-
less; and that is the whole purpose of differentiation in ordinary
microtechnique.

Benda ^^ long ago distinguished oxydirenden from einfach
losenden differentiators of mordant dyes. Einfach is not a happily
chosen word, yet the distinction is on the whole a useful one.
Potassium permanganate has been used as an oxidizing differentia-
tor, and ordinary bleaching agents are also available for the pur-
pose. The dye is presumably oxidized to a colourless substance.
The effect is one of differentiation, because certain objects contain
so much dyed matter that they still hold plenty of it when the
background has lost all visible trace. It has also been suggested ^^^
that oxidizing differentiators may act on the metal of the mordant.
Chromium trioxide has been used as a differentiator of iron
haematein.^* Here one cannot be certain of the relative roles played
by oxidation and acidity. The same applies to picric acid, which
is a moderate oxidizer and a weak acid. It differentiates iron
haematein slowly. ^^'

Another oxidizing differentiator is potassium ferricyanide. This
is used after tissues have been mordanted with potassium di-
chromate and then dyed with haematein. The technique was intro-
duced by Weigert ^^^ for the colouring of myelin, and subsequently
perfected as a histochemical test for phospholipids. ^^^'^^ The
mordant is anionic chromium, which acts very differently from the
cationic complexes that we have been considering in this chapter.

THE INDIRECT ATTACHMENT OF DYES TO TISSUES 223

The attachment of the metal to phosphohpids has been discussed
in the part of the book that deals with fixation (p. 130). The
oxidizer seems to act, partly at least, upon the fraction of the haem-
atein that has been taken up by the tissues directly, not in the
form of a lake. This technique does not provide a typical example
of the action of an oxidizing differentiator on a lake.

From what has been said it will be clear that a mordant is a salt,
the metal of which can combine with certain tissue-constituents
and can also be held in the chela-grip of certain acid dyes. It will
have suggested itself to the reader that a converse to a mordant
might exist, an acidic substance that could be taken up by certain
tissue-constituents and could also be linked to basic dyes. Such
'converse mordants' do in fact exist. Many basic dyes that are
familiar in microtechnique are used in this way in the textile
industry. Tannic acid is often used as intermediary between fibre
and basic dye. Cotton and linen have a remarkable power of taking
up this acid from aqueous solutions, and they can subsequently be
coloured by basic dyes, for which they have no direct affinity. The
compound between tannic acid and a basic dye has not, however,
the chemical complexity of a lake. The word 'mordant' loses some
of its meaning if used to include tannic acid and similar substances.
There is a suggestion of similarities that do not exist, even in
converse form.

Tannic acid and similar intermediaries are only occasionally
used in this way in microtechnique. The reason is this. The tissues
consist as a general rule of an amphoteric background with acidic
(basiphil) substances (especially chromatin) distributed in it in
the form of separate objects. One first colours these separate
objects with a basic dye or lake, and then the background with an
acid dye. The value of a lake is that it is insoluble and does not
dissolve while the acid dye is acting. A converse to a lake would
only be useful if the separate objects in cells were basic (acidophil)
and we wished to dye them with an insoluble substance before
colouring the background with a basic dye.

Tannic acid is occasionally used in quite a different way, to
prevent the escape of a basic dye that has already attached itself to
an acidic object. Any dye that began to dissolve out would at once
be precipitated. A blood-smear dyed with methylene blue/eosin,
for instance, may be treated with a solution of tannic acid,^^^ and

224 DYEING

this will retard or prevent the subsequent loss of methylene blue
from chromatin. The dye attaches itself in accordance with its own
electric charge and those of the tissue-constituents, and the tannic
acid merely traps it. There is here a strong contrast to the action of
mordants. When a mordant is used, the distribution of the colour
in the finished preparation depends on the affinity of the various
tissue-constituents for the metal.

The use of iodine by Gram ^^^ to hold gentian violet in certain
bacteria is the most celebrated example of the use of trapping
agents in microtechnique. The method was invented by the
Danish pathologist in 1884, by accident. It was his intention to
introduce a double-colouring technique for diseased kidneys con-
taining casts {Harjicy Under n) in the tubules. He intended that
chromatin should be blue with gentian violet and the casts brown
with iodine dissolved in potassium iodide solution. Gram's hope
was not realized, for the dye disappeared quickly from the sections
on subsequent treatment with alcohol. Luckily for the cause of
bacteriology, however, he decided to find whether the dye would
again be quickly lost if the same method were applied to other
organs, and he chose some that were infected with bacteria. The
result was startling, for the bacteria were intensely dyed by the
gentian violet, while all the tissue-constituents of the host organism
lost every trace of blue in the alcohol used for differentiation. Thus
the bacteria were rendered more easily visible than had previously
been possible.

Gram found that only certain particular kinds of bacteria lost
their blue colour in the alcohol, and this fact became the basis of
an important technique for distinguishing bacteria as Gram-
positive and Gram-negative. The method is still used in a slightly
modified form to the present day. Crystal violet is usually sub-
stituted for gentian violet, which is a variable mixture of the former
with related dyes. Another dye, of a contrasting colour, is often
used subsequently. This tends to disguise or displace the dye
trapped by iodine, and the term 'true Gram-positive' is sometimes
restricted to those bacteria or other objects that retain the colour
of the first dye when the second has been applied, and when 95%
alcohol has been allowed to act subsequently for a certain period.
There is, however, a large subjective element in all this, for the
periods in the various fluids are arbitrarily chosen.

Most basic dyes can be substituted for gentian violet in Gram's
technique, provided that a second dye is not used subsequently.

THE INDIRECT ATTACHMENT OF DYES TO TISSUES 225

Only eight are known, however, that give a satisfactory 'true
Gram-positive' reaction, and these are all triarylmethanes.*^' ^'^ It
w^as formerly held that pararosanilines were suitable, while
rosanilines were not.^^^ It may be recollected that rosaniline has a
methyl group attached to one of the three ar}d rings, while
pararosaniline lacks this (p. 159). Dahlia, however, is one of the
eight dyes that give a satisfactory 'true Gram-positive': yet this is
one of the rosanilines, for it possesses the methyl group.

Gram-positive and Gram-negative bacteria can be distinguished
by the use of crystal violet alone, without any trapping agent. *^
The differentiation in alcohol is so difficult, however, that the
technique is not suitable for routine use in the bacteriological
laboratory. It seems that iodine is used because it is extremely
convenient rather than because it is theoretically necessary.

There is very strong evidence that the substance that retains
crystal violet in Gram-positive bacteria is a ribonucleopro-
tein.^^^' 226, 48 j^ j^^y \^Q mentioned, however, that this is not
undisputed. ^26 j^ jg certain that a positive Gram reaction does not
always denote the presence of RXA. Indeed, one would not expect
this to be so, for cr}^stal violet will behave like other basic dyes and
is likely to be held by iodine wherever its affinity for acidic objects
has caused it to be present in particularly large amount. In the
spermatozoon of Ascaris, for instance, there is a large cytoplasmic
inclusion, the 'refringent cone', which is an object consisting of
highly acidic (basiphil) proteins. This naturally takes up a lot of
crystal violet and is strongly Gram-positive. ^^2 it contains no RNA
whatever.

The use of iodine to trap gentian violet in chromatin and thus
allow slower differentiation in alcohol was introduced by Her-
mann, 228 whose technique involved the use of another dye as well.
Gentian and crystal violets, trapped by iodine, are much used in
modern chromosome studies.^^"' 247,278,39 ^j^g method is valuable,
for the cytoplasm is of glassy transparency, while the delicate
chromosomal threads of early meiosis retain the dye.

Iodine is the most familiar trapping agent. It is seldom used in
its blue, molecular form, but is nearly always dissolved in aqueous
potassium iodide solution and thus presents itself as brown potas-
sium tri-iodide, KI3. Various other trapping agents are available,
such as bromine, mercuric iodide, mercuric chloride, potassium
permanganate, and picric acid.^^^' •*" In vital studies, as we shall
see (p. 294), ammonium molybdate is used to trap methylene
p

226 DYEING

blue. It is characteristic of all trapping agents that they precipitate
certain dyes from aqueous solutions, and that the precipitated
material has a low solubility in alcohol. Thus crystal violet is
freely soluble in 95 % alcohol, but the crystal violet/potassium tri-
iodide precipitate will only dissolve at concentrations up to 0-07%.
The crystal violet/potassium permanganate precipitate is even less
soluble (up to 0-02%). All basic dyes that have been tested are
precipitated by iodine, but a few of the precipitates are fairly
soluble in 95% alcohol. Thus the neutral red precipitate dissolves
at 0*43% and the rhodamine B at o-6i %. These dyes would not be
selected for work with trapping agents. Very few acid dyes are
precipitated by iodine. Acid fuchsine and aniline blue WS are
exceptional in this respect. ^^' *"

Potassium permanganate traps crystal violet so effectively that it
cannot take the place of iodine in the Gram technique, for it holds
the dye even in Gram-negative bacteria. ^^

The way in which trapping agents work has not been fully
established. Two possibilities present themselves. On one hand it
may be that the basic dye attaches itself as usual to acidic objects
and remains there subsequently in the presence of an extracting
agent (ethanol) simply because iodine is also present, which in-
stantly precipitates any dye that begins to be extracted. On the
other hand it is possible that an object/dye/iodine complex is
formed, which is not easily split by alcohol.

If the first possibility is true, the continued presence of iodine
all round the object is obviously necessary. The Gram method
depends upon the fact that iodine can enter Gram-positive
bacteria in the form of potassium tri-iodide, but its outward
diffusion in the form of iodine/ethanol (which is also brown) may
be hindered by factors that did not prevent its entrance. It may
be that Gram-positive bacteria have an external part, perhaps a
special membrane or cell- wall, ^^ that hinders the escape of
iodine/ethanol. The density and permeability of the Gram-
positive substance itself may also affect the escape of iodine. If
iodine can escape, the dye will be extracted by alcohol.

On this view a bacterium could be Gram-negative because the
dye could not enter it, or because there was little or no acidic
material in it to hold a basic dye, or again because there was no
special cell- wall or other feature having the property of hindering
the escape of iodine. It is noteworthy in this connexion that
if Gram-positive bacteria be crushed, they appear Gram-

THE INDIRECT ATTACHMENT OF DYES TO TISSUES 227

negative, ^^^ presumably because the iodine is free to escape into
the alcohol. If 0-25% of iodine be added to the alcohol, the ex-
traction of the dye from Gram-negative bacteria is prevented and
they therefore appear to be Gram-positive.^^'^

If this first possibility is true, it follows that iodine does not
influence the affinity of the dye for the objects it colours, but only
prevents its subsequent escape.

If the second possibility is true, the object/dye/iodine complex
may have special properties of its own ; that is, it may not only be
less easily split by ethanol than the object/dye compound, but may
also have special affinities, so that the objects coloured will not be
exactly the same as those coloured by the dye alone. This appears
to be the opinion of Panijel,^^- who has made a careful study of
crystal violet trapped by iodine. We have, however, no knowledge
of the chemical reactions involved, if indeed the triple complex is
actually formed.

CHAPTER 12

The Differential Action of Dyes

The purpose of dyeing in microtechnique is nearly always to
obtain contrast between the constituent parts of an object. If a dye
were perfectly diffuse in action, the whole of a section or other
microscopical preparation would be uniformly coloured by it.
This result is not produced by any dye, for certain parts of the
specimen are always somewhat more strongly coloured than others,
even by the most diffuse acid dyes. The mere production of con-
trast between the specimen as a whole and its surroundings is
seldom useful, though plankton organisms (for instance) may
sometimes be dyed with no other intention than this, w^hen the
desired end is recognition by external characters rather than study
of internal structure.

We have seen in chapter lo that the different objects in a
preparation may take up different amounts of the same dye, and
that different dyes (a typical basic and a typical acid dye, for in-
stance) may attach themselves differently to the same object; that
is to say, one dye may dye it deeply, another slightly or not at all.

It is now necessary to consider in greater detail the way in
which dyes may be used to give striking contrasts and thus exhibit
clearly the diversity of the parts of a microscopical preparation.

There may be chemical or physical reasons for the stronger
coloration of a particular object. It may be dyed strongly either
because it possesses many chemical groups capable of reacting
with the particular dye used ; or because there is a lot of colourable
matter in it per unit volume (that is to say, because it is very dense
in the physical sense) ; or because it is easily permeable by the dye
used, while other tissue-constituents are more difficult to penetrate.
In short, depth of coloration is affected by chemical affinity,
density, and permeability.

The matter is complicated, because these three factors may
either act together or antagonize one another. Thus chromatin is
chemically reactive (basiphil), dense, and permeable, and there-

228

THE DIFFERENTIAL ACTION OF DYES 229

fore easily dyed; red blood corpuscles are chemically reactive
(acidophil) and dense, but relatively impermeable and therefore
not easily dyed except by easily diffusing dyes. Also, different
dyes are differently affected by the chemical affinity and perme-
ability of the various tissue-constituents. The matter will be
analysed by a separate consideration of each of the three factors
affecting intensity of colouring.

Chemical Affinity

The simplest way of getting sharp colour contrasts is to take
advantage of different chemical affinities by using a basic and an
acid dye in succession. Not every pair of colours is suitable, how-
ever. It is an interesting fact that yellow basic dyes are scarcely
ever used. This has come about by a process of natural selection,
and no one seems to have mentioned the subject. The reason
is curious. Basic dyes are above all dyes for chromatin, and
chromatin exists in the form of separate objects in cells and never
forms a background against which other cellular constituents are
viewed. It is therefore desirable to stain it darkly, and to use a
light dye for the background. Now when the colour-receptors in
our eyes receive light near the middle of the spectrum, in the
region of the yellow and greenish yellow, they are stimulated in
such a way that the colour appears highly 'unsaturated' ; that is to
say, in this region of the spectrum there is an appearance of the
adulteration of the light by whiteness and thus the colours appear
pale, while the regions towards the ends of the spectrum are not
diluted in this way. It would never enter anyone's head, there-
fore, to stain chromosomes yellow and surround these objects on
all sides with cytoplasm stained blue, for the blue would make it
difficult to see the chromosomes. We therefore choose our basic
dyes from the regions towards the ends of the spectrum, while
we usually avoid the ends when choosing our background dyes.

When a black dye is used to colour chromatin (iron haemat-
ein, for instance), any acid dye used merely to colour the back-
ground must somewhat reduce the contrast.

It might be supposed that we only needed one basic and one
acid dye. It is true that we could dispense with many that are used,
without detriment; nevertheless we could not limit ourselves to
two. Differences of chemical affinity among acid dyes and among
basic dyes account in part for this fact, though differences in
capacity to penetrate are more important (p. 234).

230 DYEING

A good example of a difference in chemical affinity among dyes
is seen in the colouring of cellulose cell-walls. Most acid dyes
have no affinity for these: they act chiefly by the linking of their
anions to the amino- and other basic groups of proteins. As we
have seen, however (p. 199), Congo red and some other dyes are
able to act in quite a different way by forming hydrogen bonds with
the hydroxyl groups of cellulose.

Certain basic dyes used in pairs exhibit small differences in
affinity that can result in very noticeable differences in effect. The
most familiar example is the mixture of methyl green (triaryl-
methane) and pyronine G (xanthene). This was introduced by
Pappenheim,^^*' ^^^ who showed that the basiphil cytoplasm of
certain leucocytes could be coloured red with pyronine, while
chromatin became green or bluish green. Thus two basic dyes
coloured two basiphil substances differently. The method was
improved by Unna,^^^ whose formula included phenol. There is no
full and universally accepted explanation of the results obtained
with Unna's mixture and its modern variants, but the following
facts are relevant.

It will be remembered that if a basic dye be used at various
levels of pH, certain tissue-constituents show themselves capable
of combination with it even in very acid solution, while others are
more sensitive to acidity and react with the dye only at somewhat
higher pH (p. 194). Now if two basic dyes differed somewhat in
their capacity to bind themselves to objects at a particular pH, it
should be possible for a mixture of them to give differential
colouring.

If the usual mixture of the two dyes be used at pH 1-5, the
pyronine predominates everywhere over the methyl green; the
converse is true at pH 9-3. At intermediate pH both dyes act, but
not equally on all tissue-constituents.^^^' ^^^ One sees now how
Unna hit on the use of phenol by empirical experiments, for it
gives the weak acidity that allows both dyes to act, and to act
differentially. It is usual nowadays to buffer at pH 4-7 or 4-8.^^' ^^^

At the appropriate pH, chromatin is coloured mainly by the
methyl green and appears green, blue-green, or blue, while nucleoli
and basiphil cytoplasm are red with pyronine. It is usually RNA
that binds the red dye, but this must be confirmed by failure to
colour after the use of ribonuclease. (For a very convenient source
of this enzyme, see Bradbury.^') Similarly, reliance must not be
placed on methyl green as an indicator of the presence of DNA, for

THE DIFFERENTIAL ACTION OF DYES 23 1

as a basic dye it has general affinity for acidic tissue-constituents.
Some objects that do not contain DNA have a very marked
affinity for this dye. Certain globules in the 'vitelline' glands of the
liver-fluke, Fasciola, are an example.*^* Attempts to raise this dye
to the status of a histochemical reagent are misplaced. For a full
discussion of this subject, see Sandritter.^^^

It is claimed ^^^' ^"'^ that pyronine has a strong affinity for nucleic
acids that are depolymerized and methyl green for those that are
highly polymerized, and that the distinctive reactions are due to
the fact that RNA occurs in the tissues in a feebly polymerized
and DNA in a highly polymerized form. It is not known why the
two dyes should differ in this respect, if in fact they do. It is to be
remarked that in the absence of pyronine, methyl green will
colour the chromatin in tissues that have been subjected to a
sufficient degree of acidity to depolymerize DNA.^^^

Certain dyes are metachromatic ; that is to say, they are capable
of imparting one colour to certain objects and another to others.
This forms an important distinction between one dye and another;
but the difference is not exactly one of affinity, and metachromasy
will therefore be considered separately (p. 243).

Density

Authors often say that the cytoplasm of a particular cell is 'dense',
but in fact it is very difficult to find out how much matter
there is in a microscopical object. We can tell that a nucleolus is
denser than the nuclear sap if we see it fall under the influence of
gravity in a living cell,^^^ but this kind of opportunity seldom
presents itself. Phase-contrast may help us, but there are plenty of
traps for the unwary.*^ A study of the Becke line effect followed
by the use of the interference microscope is the surest guide, but
the actual measurement of the refractive index of microscopical
objects is not in any circumstances easy. No information on the
subject of density can be obtained by simply noticing the depth
of colouring with a dye.

In life, the greater part of the cell is made up of protein chains

intimately associated with water through -C. , -OH, -C — O,

-NH2, and other hydrophil groups. ^^° The substance resulting
from this association usually has a refractive index not very far

232 DYEING

above that of water. Ground cytoplasm commonly gives figures in
the neighbourhood of 1-353, but a less aqueous object like the mito-
chrondrial Nehenkern of an insect spermatid gives 1-376.^^^ When
fixation takes place, the water-relations of the protein chains are
entirely changed, and in a balsam preparation there is no water left.
The protein has now a refractive index of about 1-52 to 1-54. It is
precisely for this reason that we use Canada balsam as a mounting
medium: it has almost the same refractive index as the 'dry' pro-
tein, and the latter is therefore transparent. The spaces between
the protein fibres are now filled with balsam. If there was a lot of
water associated with the protein in life, these fibres will be far
apart; if there was little water, they will be close, so that there
will be more fixed tissue per unit volume. In a word, the tissue will
be dense in the strict physical sense.

Let us now imagine the dyeing of two fixed tissue-constituents,
the one consisting of a greater length of protein chain per unit
volume than the other (and therefore denser), but both exactly
equal in the number of acidic and basic groups per unit length of
protein chain. Any dye, whether basic or acid, will necessarily be
taken up in greater quantity by the former, w^hich will appear
darker in the finished preparation. There will, however, be no
obvious indication of the cause of the uptake of more dye. It
might equally well have been due to an entirely diflferent cause:
not to any difference in density, but to the fact that the protein was
(for instance) particularly acidic, and therefore bound to a lot of
basic dye.

Metaphase chromosomes are genuinely denser than the sur-
rounding cytoplasm, and the depth of their colouring is partly
due to this. One would expect them to take up basic dyes strongly,
but the protein constituent of the nucleoprotein can also be much
more strongly coloured than the cytoplasm by acid dyes. In the
same way the chromatin of the interphase nucleus can be much
more deeply coloured than the nuclear sap by acid dyes. Indeed,
deliberate use is made of acid dyes to show chromatin in certain
techniques. It has already been mentioned (p. 205) that acid
fuchsine was used for this purpose by Mallory,^^^ and azocarmine
by Heidenhain.^^^ The colouring of the protein of chromatin by
acid dyes is made easier if the nucleic acids are first eliminated by
enzyme action. Ribonuclease and DNA-ase both help the sub-
sequent action of acid dyes in colouring the protein of nucleo-
protein. ^^^

THE DIFFERENTIAL ACTION OF DYES 233

An interesting though compHcated example of rehance on
density for differential colouring is provided by Taenzer's method
for showing elastic fibres. (The method is usually called Unna's,
but Unna himself ^^^ attributed it to his pupil.) The dye used is
orcein (p. 185), dissolved in strongly acid alcohol. Differentiation
is carried out in alcohol, usually strongly acidified. The brownish-
red colour is better retained by elastic fibres than by other tissue-
constituents. The explanation appears to be as follows. *^'^

Orcein is used in alcoholic solution because it is scarcely soluble
in water. It has no particular chemical affinity for elastin. It is an
amphoteric dye (p. 190). In neutral solution (40% ethanol), being
now itself acidic, it colours collagen more strongly than elastin.
On the more acid side of its iso-electric point (about pH 5-7), it is a
feebly basic dye. It now no longer colours collagen strongly, on
account of the mutual repulsion of the positive charges, but is
taken up by various negatively charged objects. It diffuses easily if
dissolved in 70% ethanol, and is thus able to enter tissue-constitu-
ents that would have been impermeable to it in weaker ethanol.
Elastic fibres, the matrix of cartilage, acidic mucus, and chromatin
are coloured rather strongly, other tissue-constituents feebly.
Orcein is not sufficiently basic to have a particularly strong affinity
for the three last-mentioned substances, which are more electro-
negative than elastin. When the section is subsequently put in
acidified ethanol, the dye is easily extracted because it is extremely
soluble, and it would eventually be washed out everywhere.
Density rather than electric charge now^ controls events. Since
there is a lot of matter in the elastic fibres, a lot of dye is held by
them ; and when the somewhat less dense constituents (chromatin,
etc.) have lost all visible remnants of it, enough still remains in the
elastic fibres to show them clearly.

If this explanation be correct, the result is achieved partly be-
cause elastin is even denser than chromatin, partly because orcein
is too feeble a basic dye (at the pH at which it is used) to allow the
final result to be controlled primarily by electric charges.

The dyeing of elastic fibres by orcein is probably not controlled
only by their density: permeability is likely to play a part.

Carminic acid, another amphoteric dye, behaves rather similarly
to orcein; but much more of it is taken up by the chromatin, pre-
sumably because the dye is more strongly basic at the pH of the
solution. During the differentiation in acid alcohol the dye is
therefore retained as long by chromatin as by elastin.

234 DYEING

Permeability

The permeability of the various tissue-constituents plays a more
important role than chemical affinity in determining the differential
action of acid dyes in microtechnique. This fact was clear to the
genius of Ehrlich right back in 1879.

It has already been mentioned (p. 193) that Ehrlich had dis-
covered a striking fact about the granules of certain leucocytes
(eosinophils), namely, that they have a special affinity for acid
dyes. These were what he called the 'a' granules. He observed
that the *p' granules of certain leucocytes of the rabbit were also
acidophil. Leucocytes of this second kind do not occur in man.
Ehrlich drew a distinction between those acid dyes (eosin among
them) that diffused rapidly, and those (such as nigrosine and in-
duline) that diffused slowly. The eosinophil leucocyte could most
easily be distinguished from that containing the p granules by
using eosin in a single solution with nigrosine or induline. He
found that the mixture showed the a granules in the colour of the
rapidly diffusing dye, and the p granules in that of the slowly
diffusing. It was for this reason that he named the former kind
eosinophil.

'A consequence of this consideration', he wrote, 'is the hypo-
thesis that the a granulations are of closer texture (dichter) than
the p granulations; that is, that in the former the groups of
molecules {Micellen of Naegeli, Syntagmen of Pfeffer) are
larger and the intermicellar spaces smaller than in the latter.
. . . The molecules of the easily- diffusing eosin penetrate much
more quickly into the narrow osmo-regulatory spaces of the a
granulations than those of nigrosine, which diffuses with
difficulty; and so the micelles of the granulations are already
saturated with eosin before the second dyestuff can enter them
at all. In contrast to this, the molecules of nigrosine can enter
the wider intermicellar spaces of the p granulations and so
achieve an important colour-effect.' ^^^

Thus Ehrlich showed that while differential dyeing was in some
cases caused by the chemical differences between basic and acid
dyes, in others it was due to physical factors in dyes and objects.
At the time he was 24 or 25 years old (fig. 24, opposite p. 193).

The differential action of the rapidly and slowly diffusing dyes
is reflected in their industrial use. The professional dyer classifies

THE DIFFERENTIAL ACTION OF DYES 235

the acid dyes according to the way in which they can best be used to
colour textile fibres. ^^^''^'^^^'^^^ It is desirable to mention this
classification, partly because one can only follow the textile litera-
ture if it is understood, partly because it has direct significance for
microtechnique. The classification is not based on chemical re-
lationship: dyes in a single group may possess quite different
chromophores. The limits of some of the groups are not sharp, and
the textile authorities do not all use exactly the same classification.
Three groups will be defined here.

The 'levelling' dyes are acid dyes that are used to colour wool
and other protein fibres from a strongly acid bath. They have not
a very high affinity for such fibres and will not dye them at neutral-
ity, and they have no affinity for cellulose fibres. The word 'level-
ling' (or 'equalizing') means that they dye very evenly. They are
often called simply 'acid dyes', ^^^'^^^ in reference to the pH of the
bath in which they are dissolved, but this confusing name will not
be used here.

The 'milling' dyes colour protein fibres very strongly at low pH,
but their action is so uneven that they are not used in this way in
practice. They are dissolved, on the contrary, in neutral or weakly
acid solution and are therefore sometimes called 'neutral- dyeing'
dyes. Their special character is that they are not decolorized nor
extracted by 'milling', which is a felting process applied to wet
wool after dyeing, often in the presence of soap. It is characteristic
of milling dyes that they are of greater molecular weight than the
levelling ones, and form colloidal solutions. ^^*

The 'direct cotton' dyes colour cellulose fibres without the use of
any mordant.

The relevance to microtechnique of this classification of acid
dyes will appear shortly.

Acid dyes are very often used in pairs. Sometimes the dyes are
mixed together, sometimes one is used after the other. If the
affinities of the two were exactly the same, no advantage would be
secured, for the appearance given would be exactly the same as
though only one dye had been used (apart from the colour being
mixed). In fact, however, the dyes are selected in such a way that
certain objects are coloured by one of them, others by the other,
and some (usually) by both. Sometimes three acid dyes are used.

One component of the pair (or trio) colours collagen fibres,
another the ground cytoplasm. Other tissue-constituents are
coloured by the one or the other or by both, but for our present

236 DYEING

purpose it will suffice to concentrate on collagen and cytoplasm.
The dyes that colour the cytoplasm from these pairs are mostly the
ordinary background dyes used for contrast with the basic dyes
that colour chromatin. The most typical of these are eosin
(xanthene), orange G (azo), ponceau 2R (azo), and especially
picric acid (nitro). It is interesting to notice that most of the dyes
used in microtechnique for colouring the background are levelling
dyes.

The most typical of the dyes for collagen are aniline blue WS
and the closely related methyl blue (triarylmethane), induline WS
and nigrosine W (azine), diamine blue 2B and naphthol black B
(azo), and indigo-carmine (indigo dye). The striking fact about
this apparently random set of dyes is that none of them belongs to
the 'levelling' group. Methyl blue, for instance, can be used for
cotton, and is indeed sometimes called 'cotton blue'; diamine
blue 2B is a direct dye for cotton; and others in this group, to a
greater or lesser extent, have the characters of milling dyes.

In brief, then, the feeble levelling dyes colour the cytoplasm, the
vigorous milling and cotton dyes colour collagen. Why?

The first clue was obtained in the twenties by a Frenchman,
Collin, ^2^' ^^^ who made a special study of Mann's methyl blue/
eosin. ^^^' ^^^ He dissolved gelatine at various concentrations in
warm water, dipped microscopical glass slides in these solutions,
dried the films thus produced, and fixed them in a mixture of
formaldehyde and alcohol. After washing and again drying them
he put them in Mann's mixture. The films made from concen-
trated solutions of gelatine took up the red colour of eosin, those
from weak solutions the blue of methyl blue.

These results suggested that methyl blue was not able to enter
concentrated gelatine, but that wherever it could enter and com-
pete with the eosin, it dominated the latter. In other words, the
blue dye was more vigorous in action, but penetrated with
greater difficulty than the red.

Collin now showed that eosin penetrated much more rapidly
than methyl blue into gelatine gel contained in a test-tube, and
also that if Mann's mixture was put in a collodion sack, and the
sack in water, the water was coloured by eosin before the methyl
blue escaped. A solution of methyl blue, filtered, contains particles
that are visible under the microscope: a solution of eosin does
not. Various objects (blotting-paper, animal charcoal, etc.) take
up much more methyl blue than eosin from solutions at the

THE DIFFERENTIAL ACTION OF DYES

237

same concentration. The blue dye is well retained, the red easily
washed off.

MoUendorff ^^^ reached essentially the same conclusions from
independent studies. As he put it, from mixtures of two acid dyes,
the more diffusible goes into the more compact structures, the
more colloid into the more pervious.

This subject was subsequently investigated in detail by Seki,
who noted the rate of penetration of dyes into agar gels contained
in test-tubes. In his experiments, of which full particulars are given
in his paper,^^^ the distances penetrated by certain acid dyes in 15
hours were these: —

picric acid .
acid fuchsine
nigrosine W
methyl blue,
aniline blue WS

25 mm
10

7
5
4

)5

J>

J>

>)

Details of a similar experiment are given in the Appendix (p.
322). The distances penetrated in 48 hours were as follows: —

orange G
methyl blue .
aniline blue WS

37 mm
16

12

n

))

It will be noticed that the dyes that penetrate quickly are the
ones that colour the cytoplasm and those that penetrate slowly are
the ones that colour collagen, when suitable pairs are used.

The fact that we are not concerned here with special chemical
affinities, but rather with the factors that have been mentioned, is
shown very clearly by the behaviour of acid fuchsine. This is an
intermediate kind of dye, for it is level-dyeing, yet moderately fast
to milling,^^^ and it penetrates at moderate speed. In Mallory's
technique ^^^ it is used with anihne blue, and here it colours the
cytoplasm; but in various techniques, of which Hansen's ^^^^ is
an example, it is mixed with picric acid, and now it colours the
collagen. It is clear that we are not concerned here with specific
affinities, but with the relative positions of the various dyes in a
scale of characters. The extremes in this scale are aniline blue WS
and picric acid.

The dyes used for collagen mostly have higher molecular
weights than those used for cytoplasm (e.g. methyl blue 800,
picric acid 229). This, however, is not the direct cause of the

238 DYEING

difference in rate of penetration. Many dyes are dispersed in
particles that are larger than single anions. Estimates of the size of
the particles can be obtained by measurement of osmotic pressure
or electrical conductivity or by ultra-centrifuging or ultrafiltra-
tion through cellophane or collodion film.*^^''^^ It is clear that
the anions of a dye often aggregate together. Sometimes the par-
ticles are formed by the aggregation of several anions only, some-
times by the aggregation of several anions with a smaller number of
sodium or other cations, the remaining cations being free. The
resultant charge on the dye particles of an acid dye therefore varies
in different cases, though it is always negative. It is probable that
ion-aggregates break up below the temperatures at which textile
dyeing takes place, but they are present at room temperature and
play an important part in microtechnique. The dyes used for
collagen are the ones that form large aggregates, while the typical
background or levelling dyes are dispersed as single ions.

The size of the spaces or pores in the constituent parts of fixed
microscopical preparations is not known, but the structure of wool
may give us some impression of what to expect. The protein is
here in the form of long, nearly parallel chains. Here and there the
chains become quite parallel and closely bound together, so that
a submicroscopic crystal or micelle is formed. The chains emerging
from the end of a micelle wander loosely for a bit before they enter
and form part of several other micelles. Thus there are minute
crystalline and non-crystalline regions in the fibre, the former too
compact to be entered by any dye, the latter loose and containing
spaces between the somewhat irregularly arranged threads. The
available evidence suggests that the diameter of these spaces is
about 3 J or 4 m/x.^^^ Dyes must enter these if they are to permeate
and colour the fibre. The cellulose fibres of cotton are arranged in
a very similar way, the spaces being about 2 to 10 m/x in diameter.

It is probable that the various constituents of a fixed micro-
scopical preparation vary greatly in the size of the spaces within
them.*^^'*^^ Collagen is an example of a substance of very loose
texture, readily entered by any dye; cytoplasm has a tighter con-
sistency and is more selective towards dyes; the contractile sub-
stance of muscle is somewhat tighter still; while the red blood-
corpuscles of mammals are among the least pervious of all tissue-
constituents. The dyes that colour red blood-corpuscles are those
that diffuse particularly easily through a fine collodion membrane.
With three selected acid dyes one can colour collagen, ordinary

THE DIFFERENTIAL ACTION OF DYES 239

cytoplasm, and red blood corpuscles in three different colours;
for instance, with aniline blue, acid fuchsine, and orange G
respectively in Mallory's technique.

The fact that it is so particularly easy to colour collagen and red
blood corpuscles differently is interesting. Both of these are ex-
amples of strongly basic substances, for collagen contains a high
proportion of arginine and lysine, and the globin of haemoglobin is
rich in these and in histidine. One would therefore expect them
both to be strongly acidophil. So indeed they are; but red blood
corpuscles are so impermeable that the most powerful milling
dyes can scarcely enter them. Thus in the differential action of
acid dyes, physical or mechanical factors predominate over
chemical affinities.

Some of the powerful but slowly diffusing dyes used for collagen
colour collodion in acid solution. *^^ Thus aniline blue and methyl
blue dye it strongly from pH2 to PH5. This is unusual behaviour
for acid dyes. One would expect the negatively charged anion to
be repelled by the similarly charged substrate. These two dyes
have some capacity to act as though they were basic, and indeed
aniline blue possesses an amino-group. Once again acid fuchsine
is intermediate between these and the levelling dyes, for it colours
collodion moderately from pHz to pH6.

Induline colours collodion less strongly at low pH, because it
tends to flocculate.

When it is desired to colour collagen differently from other
tissue constituents, use is often made of phosphomolybdic acid.
The techniques employed are variants of the procedure intro-
duced in 1900 by the American histopathologist, Mallory.^^^ In
his technique sections were treated with an aqueous solution of
phosphomolybdic acid and then with a mixture of aniline blue,
orange G, and oxalic acid in water. The oxalic acid served simply
to lower the pH and thus help the action of the levelling dye,
orange G. (The dyeing of the chromatin is irrelevant and will not
be considered here.)

Molybdenum is a metal related to chromium. The yellowish
white oxide, M0O3, insoluble in water, dissolves in ammonia
solution to produce ammonium molybdate (p. 294), which reacts
with orthophosphoric acid, to produce ammonium phosphomoly-
bdate; this, when dissolved in aqua regia, deposits pale yellow

240 DYEING

crystals of phosphomolybdic acid. The composition of these
crystals is not quite constant, but approximates to H3P04(Mo03)] 2,
with water of crystallization.

The function of phosphomolybdic acid in Mallory's and similar
techniques was explained by the researches of Mollendorff ^^^ and
Seki.461

If an Irish bull be permissible, one may say shortly that phos-
phomolybdic acid acts as a colourless acid dye (for it scarcely
colours the tissues). Luckily one can convert it into a coloured
substance by exposure to bright light. A blue lower oxide of moly-
bdenum, of indeterminate composition, is produced. If a micro-
scopical section be soaked in a solution of phosphomolybdic acid
and then exposed to light, the blue colour reveals that it was
present chiefly in the collagen; much less in the cytoplasm; less
again in muscle; and least of all in red blood corpuscles. This is
exactly the same distribution as is shown by methyl blue, and
phosphomolybdic acid thus acts as though it were a very slowly
diffusing acid dye. The anion is large, and its size is increased by
hydration.

If a section be treated with phosphomolybdic acid and then with
one of the background or levelling dyes at low concentration in
the presence of the same acid, the background dye will colour
nothing except the red blood-corpuscles. Thus the phospho-
molybdic acid acts as a dye-excluder towards the background dye.
One is reminded of the use in the textile industry of 'resists', or
substances that prevent the subsequent action of dyes.''^ A mixture
can be made of normal and 'resisted' wool, and this gives a varie-
gated effect when dyed. Some of the substances used are colourless
sodium sulphonates, which act very much as though they were
colourless dyes.

When a section is first treated with a typical background dye,
or with some other dye (such as acid fuchsine or azocarmine) that
diffuses more easily than aniline blue, and then with phospho-
molybdic (or phosphotungstic) acid, the latter competes with the
dye wherever it can enter. It enters the collagen most easily. If
the treatment be stopped at the right moment, the dye is turned
out of the collagen, but left in the cytoplasm, muscle, and red
blood-corpuscles. If now the section be rinsed and treated with
aniline blue or a similar dye, the collagen will be coloured ex-
clusively by this.

The treatment with phosphomolybdic acid also helps differ-

THE DIFFERENTIAL ACTION OF DYES 24I

ential colouring in another way. In so far as it enters the cytoplasm
and muscle and is taken up by them, it helps to exclude the aniline
blue, and thus appears to favour the background dye. All these
processes, however, have to be carefully controlled. If the aniline
blue or similar dye is allowed to act for too long, it will spread to
the cytoplasm and muscle and eventually replace the background
dye.

It is particularly to be noticed that the phosphomolybdic acid
opposes the action of the aniline blue. One sometimes sees state-
ments to the effect that it 'mordants' the tissues for the aniline
blue. Not only is it impossible for such a substance to mordant for
an acid dye, but in fact the aniline blue colours everyAvhere more
powerfully if the treatment with phosphomolybdic acid be
omitted. To prove this it is only necessary to take two sections
from the same block, put one of them in a solution of phospho-
molybdic acid, and then colour both sections with aniline blue for
the same period.

An object that is difficult to penetrate will resist the escape of a
dye that has succeeded in entering it. One may take a dye that does
not diffuse readily, heat it until the ion-aggregates have dispersed,
allow it to enter the tissues in this form, and then cool the dyed
object: the dye is unable to escape. This process is much more
applicable to textiles than to microscopical preparations, for the
former are nearly always dyed at high temperatures. Polar yellow
R, for instance, will not enter wool at all below 40° C, because the
ion-aggregates are too large. ^^^ Similarly one can scarcely colour
mitochondria strongly with cold acid fuchsine solution, but the
dye enters them readily when the temperature is raised to near
boiling point. If subsequently the section be treated at room-
temperature wdth another acid dye, even a readily- diffusing one,
the acid fuchsine will be replaced in the cytoplasm before it leaves
the mitochondria. This is probably the basis of Metzner's ^*^ and
several other methods for mitochondria, in which acid fuchsine is
used hot and another dye (or dyes) at room-temperature sub-
sequently. In Altmann's original method,^ hot acid fuchsine was
followed by warm picric acid. He himself admitted that the
differentiation was difficult. It is far easier to use cold picric acid
solution, as Metzner ^*^ did. (See also Meves ^^^ for details of
Metzner's method.)

That this is the correct explanation of the usual mitochondrial
Q

242 DYEING

methods is suggested by the fact that red blood-corpuscles
generally retain the acid fuchsine in such preparations. Mito-
chondria can also be coloured by the basic dye, crystal violet,
used hot.^^' ^^

Chemical affinity, density, and permeability play their allied or
antagonistic parts in the colouring of tissue-constituents, and it is
difficult to disentangle their effects. If, in any particular case, we
can be sure that there is no obstacle to penetration, and if we know
the chemical constitution of the substance that reacts with the
dye, and if further the substance is chemically homogeneous or
nearly so (unlike most proteins as they occur in the cell), we may be
able to estimate the amount of the substance present by measuring
the optical density of the dye taken up by it.^^-^ Thus a basic dye
may be used, at a pH too low to colour protein, to obtain an
approximation to the amount of DNA in a nucleus or of RNA in a
nucleolus or in the cytoplasm. A mordant-dye, chrome alum/gallo-
cyanine (p. 215), is particularly recommended for this purpose. *^^

CHAPTER 13

Metachromasy

The words metachromatic and metachromatism were intro-
duced in 1876 ^' ^ in reference to the changes in colour undergone
by certain substances when heated. The adjective, however, is used
in biology in a different and very special way. The corresponding
noun is metachromasy or metachromasia.

If a section is dved with toluidine blue, manv tissue-constituents
will be coloured blue, but if there is any cartilage in the preparation
its matrix will be dyed purple or red. It might be thought that the
dye was impure, owing to faults in manufacture or subsequent
changes in chemical composition, but the effect is observed
equally well with pure specimens of the dye. This is a typical
example of metachromasy. Toluidine blue is said to be a meta-
chromatic dye. The matrix of cartilage is called a chromotrope:
that is to say, a substance capable of altering the colour of a
metachromatic dye. The corresponding adjective is chromotropic.
The word orthochromatic is used to mean non-metachromatic.
Thus whatever is coloured blue by toluidine blue is said to be dyed
orthochromatically, and dyes that do not give metachromatic
effects are called orthochromatic.

Certain dyes are not stable in solution, but gradually give rise to
other colouring agents, which are then present as impurities and
can be separated by suitable means. The presence of such im-
purities of spontaneous origin is called allochromasy. This has no

necessarv connexion with metachromasv, but an orthochromatic

■J • '

dye may give rise to metachromatic impurities by allochromasy.
Solutions of Nile blue are allochromatic, for they contain not only
the ions that one would expect, but also another substance, an
oxazone (p. 301); in addition to this, however, the cation of the
dye is feebly metachromatic.

Metachromasy was discovered independently three or perhaps
four times during the year 1875. The evidence suggests that
Jiirgens ^^^ was the first to report his findings publicly. He used

243

244 DYEING

dahlia in a study of amyloid degeneration and was surprised to
find that this violet dye coloured the amyloid corpuscles a brilliant
red. The celebrated histologist, Ranvier,*^^ dyed cartilage meta-
chromatically with cyanine, while another Frenchman, Cornil,^^^
used methyl violet on the same two chromotropes that were
observed by Jiirgens and Ranvier. It is possible that the Austrian
pathologist, Heschl,^^^ also saw a metachromatic effect in 1872
and published it in 1875, but this is not certain. He accidentally
dyed the skin of his fingers with some violet ink and then tried it
on various other tissues, including liver and kidney in amyloid
degeneration. The degenerate parts were coloured dark rose-red,
everything else blue. This effect may indeed have been due in part
at least to the metachromatic dye, aniline blue (spirit soluble),
which was present in the ink; but since this also contained basic
fuchsine, one cannot be sure. (In this paragraph the modern names
of the dyes have been used throughout.)

It has been stated more than once ^^^' ^^°' ^^^ that the word meta-
chromasy was introduced and defined by Ehrlich in his paper of
1877.^^^ Very remarkable things are believed about this paper, by
persons who have not read it. It has been said that he here intro-
duced the idea of classifying dyes as acid and basic,^^^ and objects
in tissues as basiphil and acidophil. ^^^ In this paper, written when
he was still a medical student, Ehrlich gives a competent account
of the form, distribution, and reaction to dyes of what were
obviously the basiphil cells (Mastzellen) of connective tissue,
though he refers to them throughout as Plasmazellen (see
Westphal ^^*). He notes the colour-change of the dye, but makes
no attempt to define metachromasy and does not use the word.
Ehrlich's first scientific paper foreshadows rather faintly his
subsequent contributions to our understanding of the action of
dyes in biological microtechnique.

In a later paper ^^^ Ehrlich remarks that certain dyes colour the
granulated cells {Mastzellen) of connective tissue 'metachromatic-
ally, that is, in a tint differing from the colour of the dye used'. It
is more accurate to define metachromasy as the colouring of differ-
ent tissue-constituents in different colours by a single dye. The
words 'single dye' must here be taken to mean that the substance
that dyes the different tissue-constituents in different colours
can be extracted from the dye-solution in dry form as one pure
chemical compound, not as two or more.

In this sense there are both basic and acid metachromatic dyes,

METACHROMASY 245

but whereas the basic ones are important in microtechnique and
all act on the same chromotropes, apparently in essentially the
same way, the acid metachromatic dyes are relatively unimportant
and act quite differently from the basic ones.

The metachromasy of basic dyes will be dealt with first.
Unnecessary repetition of the words 'of basic dyes' will be avoided.
All general remarks on metachromasy are to be understood as
referring to the metachromasy of basic dyes, unless the contrary is
clearly indicated.

Metachromasy is of importance in histochemistry, because very
simple techniques give striking results that help towards the
chemical identification of tissue-constituents.

The most obviously chromotropic tissue-constituents are the
following: —

the matrix of cartilage ;

the secretions of certain mucous glands ;

the granules of the basiphil cells (Mastzellen) of connective

tissue ;
the corpuscles of amyloid degeneration ;
the 'volutin' granules that occur in yeast and in certain

diatoms and bacteria.

Certain substances prepared from the cell-walls of various red
algae are strongly chromotropic. Agar is an example.

Various tissue-constituents other than these are also chromo-
tropic, but less strikingly so. Chromatin is an example of a weakly
chromotropic substance. All chromotropes that occur in micro-
scopical preparations are basiphil, though not all basiphil objects
are chromotropic.

The first person to study metachromasy in detail by the examina-
tion of pure substances in glass vessels was Lison,^^^ whose work
has been extended by Sylven.*^^

A large number of substances that are very familiar components
of organisms are not chromotropes. The following are examples: —
t/-ribose, sucrose, maltose, cellobiose, lactose, dextrins, glycogen,
starch, hemi-cellulose, cellulose, inulin, gums, mucilages, pectic
substances; serum-albumin, serum globulin, fibrin, collagen,
keratin, myosin, silk. No lipid is obviously chromotropic, though
cerebrosides may perhaps be feebly so. It is characteristic of
chromotropes that they are acidic, and this is of course related to

246 DYEING

the fact that the chromotropic objects seen in microscopical
preparations are basiphil.

The acidic groups present in chromotropes are sulphuric,
phosphoric, and carboxyl. As Lison ^^^ showed, most of the
familiar, strikingly chromotropic objects owe their character to
the presence of sulphuric esters of polysaccharides of high
molecular weight. It will be recollected that a sulphuric ester
differs from a sulphonic acid by the possession of an extra oxygen
atom linking the organic radicle to the sulphur. Several such com-

RO^ ^O R^ ^O

Sulphuric ester Sulphonic acid

pounds are mucosubstances. Chrondroitic acid, for instance, makes
the matrix of cartilage chromotropic ; mucoitic acid does the same
for certain mucous secretions ; heparin for the granules of Mast-
zellen. Chromotropic sulphuric esters need not, however, be muco-
substances. Agar, for instance, is the calcium salt of a sulphuric
ester of a pentose polysaccharide that lacks any amino-group.

The phosphate group does not generally confer such a strongly
chromotropic character as the sulphuric. Adenosine triphosphate is
not chromotropic, RNA only very slightly so, DNA rather weakly
(see p. 258). Some of the metaphosphates, however, are rather
strongly chromotropic. These substances tend to polymerize. The
general formula for potassium metaphosphates is (KPOg),^. When
n is very large, the substance is strongly chromotropic; when
moderate, weakly ; sodium trimetaphosphate is not chromotropic at
all. A potassium metaphosphate of rather high molecular weight
can be extracted from the mould, Aspergillus niger, and this is
strongly metachromatic. It has been shown ^^^ that the meta-
chromatic particles present in yeast {Sac char omyces cerevisiae) and
certain bacteria contain a metaphosphate associated with protein.
There seems to be no doubt that this substance corresponds to the
Volutin' of earlier authors.

The carboxyl group has less chromotropic effect than the phos-
phate. It occurs in uronic acids as a component of many muco-
substances. Some of these are sulphuric esters, and the sulphuric
component then overshadows the carboxylic in chromotropic
effect. When, as in hyaluronic acid, there is no acidic group other
than the carboxylic, the substance is only feebly chromotropic.

Other acidic radicles than sulphuric, phosphoric, and carboxylic

METACHROMASY 247

do not confer the chromotropic property. Thus nitrocellulose is
acidic and therefore basiphil, but not a chromotrope. (For the
reaction to dyes of nitrocellulose in the form of collodion, see
p. 193.)

COOH

Skeleton formula of glucuronic, galacturonic, and mannuronic acids

There is a strong correlation between degree of polymerization
and chromotropic effect. Thus glucuronic acids by themselves and
hyaluronic acid are scarcely chromotropic, while sodium alginates
(high polymers of mannuronic acid) and concentrated gels and
solid films of hyaluronic acid show a definite colour-shift.^^*
There appear to be two separate factors affecting the degree of
chromotropy achieved: the nature of the acid groups, and their
degree of separation in space. With any particular acid group, the
greatest colour-shift will be shown by a highly polymeric gel or film
crowded with sites of negative charge.*^* This crowding will natur-
ally result also in strong basiphilia towards orthochromatic dyes.

It was shown by Lison ^^^ that various non-chromotropic high
polymers occurring in the tissues of organisms, such as glycogen,
starch, cellulose, gum arabic, and chitin, can be rendered strongly
chromotropic by their artificial conversion into sulphuric esters.
Sylven *^* has extended this work by observing the gradual in-
crease in degree of chromotropy as more and more carboxymethyl
groups are introduced into cellulose.

Similar effects can be observed in microscopical preparations.
If a section be treated briefly with concentrated sulphuric acid,
any neutral polysaccharides will be transformed to sulphuric
esters and will therefore become basiphil and metachromatic."*
Glycogen and neutral mucopolysaccharides (such as the 'mucoid'
of the cells lining the mammalian stomach) give this reaction. The
same result may be achieved in a different way, by simply placing
a section in a solution of chromium trioxide.*^^' -^^^ Lison ^^^ con-

siders that a ^C. group of the saccharide component is

/ \h .0

oxidized through aldehyde to — C. , presumably with

breakage of the ring.

248 DYEING

Certain inorganic substances with complex anions are markedly
chromotropic.*^'*^^ Among these anions are ferricyanide, thio-
cyanate, and especially phosphotungstate. The subject has not
been fully investigated, presumably because it is not of much
interest to biologists, who are the chief people concerned with
metachromasy.

More than two dozen basic dyes are known to be definitely
metachromatic, most of them being triarylmethanes and azines.
No azo-dye is metachromatic except Janus green, which owes this
character to the fact that it is also an azine. Among the lake-dyes
certain oxazines are remarkable for giving strongly metachromatic
effects. ^^*' *^'^' ^^^ The most useful metachromatic dyes are prob-
ably these : —

methyl violet (triarylmethane)

brilliant cresyl blue (oxazine)

cupric, ferric, and aluminium lakes of coelestine blue

(oxazine)
thionine (thiazine)
azure A (thiazine)
azure B (thiazine)
toluidine blue (thiazine)

It will be noticed that the thiazines are pre-eminent in providing
us with valuable metachromatic dyes.

Certain dyes show their metachromatic effect when used vitally.
These will be considered in one of the chapters devoted to vital
dyeing (p. 281).

It will be convenient to consider first an ideal dye that has a
nearly symmetrical absorption curve with a peak in the middle of
the visible spectrum. Basic fuchsine w^ould serve as a fairly good
example, though the curve is not very symmetrical. Such a dye
will necessarily transmit light from the two ends of the spectrum
(compare fig. 17, p. 161), and if the transmission curve is regular
on the two sides, the colour will be purple. (Basic fuchsine trans-
mits a high proportion of red and the colour is magenta.)

If now we somehow influence our dye in such a way that the
peak of its absorption curve (or the trough of its transmission
curve) is slowly shifted towards the right (that is, towards the
longer wave-lengths), the transmitted colour will change gradually
from purple through violet to blue and then to green. The

METACHROMASY 249

colour is said to be 'lowered', and the mean wave-length of
the transmitted light has indeed been lowered. The influence
we have brought to bear is therefore said to be 'bathochrome'. If
on the contrary w^e somehow shift the peak of the absorption
curve of our purple dye towards the left, we 'heighten' the colour
through magenta to red, orange, and then yellow. The influence
has been 'hypsochrome', since the mean w^ave-length of the trans-
mitted light is now higher (longer). It is obvious that if either a
bathochrome or a hypsochrome influence were pushed far enough,
so that the absorption curve were moved right outside the visible
spectrum, the transmitted hght would brighten to white, and that
is indeed why the two ends of the scale (green and yellow) are
rather similar.

We are now in a position to lay down a general law about meta-
chromasy. The metachromatic effect is hypsochrome. This law is sub-
ject only to the exceptions mentioned on pp. 258 and 259. Colours
may be arranged in an order of increasing hypsochrome effect,
thus: green, blue, violet, purple, magenta, red, orange, yellow. The
metachromatic colour given by a dye is to the right of the ortho-
chromatic in this list. It follows that no metachromatic dye can be
yellow, for this would render chromotropes colourless. All the most
valuable metachromatic dyes are blue or violet, and the colour-
shift is generally from blue to purple or magenta, or from violet to
red. The normal human eye is very sensitive to these changes. If a
red dye had its absorption-maximum shifted by the same amount
in wavelength as one of these dyes, the change in colour would
not appear to us so striking. We seldom choose a red dye, such as
neutral red or safranine (azines), when we want metachromatic
effects.

In passing from the orthochromatic to the metachromatic
colour the peak of the absorption curve may move a particularly
long distance; or, alternatively, the colour-shift may be one that
is particularly evident to the human eye, even though the peak of
the curve may not have moved very far. Thus a dye may be con-
sidered highly metachromatic for one of two alternative reasons,
which may be called respectively objective and subjective.

The colour-shift could be pictured mentally as a bodily move-
ment of the absorption curve across the spectrum, without any
change in its form; but in fact the shift is more complex than
this.^*^' ^*^' *^^' *^^

250 DYEING

When a metachromatic dye is dissolved in absolute alcohol or
other organic solvent, the absorption curve shoves a single main
peak, called the a band. With toluidine blue the peak is at 630 mjit
or a little less, in the reddish orange. The transmission curve
naturally shows a trough, which w^ill here be called the a trough.
A glance at the transmission curves (fig. 27, a) suggests rightly

100

80 -

3?

60

in

to
Z40

20 -

400

450

500

550

600

650

WAVELENGTH, m|i

FIG. 27. Graph showing the transmission of light of various wave-
lengths through a layer of toluidine blue solution i cm thick. ^^

A, the dye was dissolved at 0-0002% in 80% alcohol; B, at 0'00i% in distillsd
water; c, the same as B, with the addition of 2 drops of heparin solution to 3*4 ml
of solution. The heparin solution used contained 5000 international units per ml.
The a, 13, and y troughs in the curves are marked by arrows.

that the colour of the solution is blue. When the dye is dissolved
in aqueous solution a second or p trough appears, corresponding
to a p band or hump in the absorption curve. It is claimed that this
can just be detected even in non-aqueous solutions. ^^^ It is not
ordinarily detectable in very dilute aqueous solutions, but becomes
more and more marked as the concentration rises, while the a
trough becomes shallower. The ^ trough is always situated on the
side of the a trough towards the shorter wave-lengths. In fig. 27,
c, the p trough is deeper than the a, and the two are almost
smoothed out into one. The wave-length of the p trough of
toluidine blue is about 590-600 m/x (yellowish orange).

METACHROMASY 251

When a strongly chromotropic substance is added to the solu-
tion, a new trough (y trough) appears, still further in the direction
of shorter wave-lengths. The addition of quite a small amount of
an intensely chromotropic substance, such as the heparin of
Mastzellen, suffices to hollow out a considerable y trough at the
expense of a and ^ (fig. 27, b). With toluidine blue the wave-
length of the y trough is about 550 m/x (yellowish green). The
colour of the solution is purple, since light is freely transmitted
from both ends of the spectrum.

Varying degrees of colour-shift will be produced by variations
in the tendency of chromotropes to flatten out the a transmission
trough and deepen the p and y. Most chromotropes produce their
main visible effect by lowering the y trough, though they lower the
P at the same time. RNA, however, a feebly chromotropic sub-
stance, makes a low p trough at certain concentrations, without
affecting the y region. ^*^

Some metachromatic dyes show quite a low trough in the
transmission curve in the region of the ultra-violet, but this is not
affected by the presence of chromotropes. ^^^

We naturally ask ourselves whether there are any features of chem-
ical composition that separate metachromatic from orthochromatic
dyes. Certain general remarks may be made under this head.

Dyes in which all the -NH2 auxochrome groups have the
hydrogen atoms replaced by -CH3 or -C2H5 are not meta-
chromatic. This applies, for instance, to crystal violet and methyl-
ene blue. These dyes are often regarded as somewhat meta-
chromatic, but there is reason to believe that they would be quite
orthochromatic if perfectly pure.^°^ The former is generally
contaminated with the highly metachromatic methyl violet, and
methylene blue always gives rise by allochromasy to the azures, so
that perfectly pure specimens have not been obtained.

Not all dyes that have an unsubstituted -NHg group are meta-
chromatic. It is a remarkable fact that the dyes that are meta-
chromatic are, in general, those that are capable of being trans-

+
formed to imino-bases. The change from an =NH2 group to an

imino (^NH) group does not involve a loss of quinonoid structure
in the ion. The imino-bases are therefore coloured, unlike the
leucobases. The chemical structure of the imino-base of pararos-
aniline, for instance, may be compared with that of the leuco-
base (p. 163). The transformation from dye to imino-base involves

252 DYEING

a hypsochrome effect, for the dye is magenta, the base reddish
orange. PararosaniHne is metachromatic, and it exempHfies the
general rule that the transformation of a metachromatic dye to its
imino-base involves a heightening of colour : that is to say, a change

NH, NH,

NH

Imino-base of pararosa?tiline

in the same direction as the metachromatic shift. Because the
colour changes when the imino-base is produced, and because a
certain degree of alkalinity is necessary for its production, it
follows that metachromatic dyes can be used as indicators
of pH.

It is most tempting to assume, as Hansen ^^^ did nearly half a
century ago, that chromotropes take up imino-bases from solutions
of metachromatic dyes. He considered that these bases were
present in ordinary solutions of the dyes and that they were some-
how specifically stored up by the chromotropic tissue-constituents.
He remarked that if one shakes up benzene, xylene, or chloroform
with an aqueous solution of thionine, the little oily droplets give
the impression under the microscope that they are composed of
'mucin', because they have the metachromatic colour.

Although there may be some roundabout connexion between
the capacity of a dye to form an imino-base with heightened
colour on one hand and its capacity to give a metachromatic effect
on the other, yet